Method of selection for treatment of subjects at risk of invasive breast cancer

ABSTRACT

The present disclosure provides compositions and methods for the treatment of subjects having a risk of invasive breast cancer. In some embodiments, these aspects allow for the pairing of the proper treatment option for the particular subject. In some embodiments, this allows for identifying subjects who, while at risk for invasive breast cancer, will not normally respond to radiation therapy, and can instead receive an alternative therapy, such as a HER2 antibody.

RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C. §371 of International Application No. PCT/US2019/051128, filed Sep. 13,2019, which claims priority to U.S. Provisional Application Ser. No.62/731,316, filed Sep. 14, 2018. The contents of each of these relatedapplications are hereby incorporated by reference in its entirety.

BACKGROUND Field

The present technology generally relates to whether or not a subject whois at risk of invasive breast cancer will be responsive to various formsof cancer therapy.

Description of the Related Art

There are a variety of markers for the identification of tumors insubjects. In addition, there are various markers that can be used forthe prediction of neoplastic progression. For example, U.S. Pat. Pub.Nos. 2010/0003189, 2012/0003639, and 20170350895 disclose a variety ofmarkers that when examined in various combinations can predict thelikelihood that a subject will have DCIS and/or invasive breast cancer.

SUMMARY OF VARIOUS EMBODIMENTS

In some embodiments, a method of treating a subject is provided, themethod comprises identifying a subject with DCIS that has an elevatedlevel of activity in a k-ras pathway; and administering an aggressivebreast cancer therapy to the subject, wherein the aggressive breastcancer therapy is not radiation. In some embodiments, the k-ras pathwayis elevated if there is an elevated level of at least one of: K-ras,RAF, MAPK, MEK, ETS or SIAH.

In some embodiments, a method of treating a subject is provided. Themethod comprises identifying a subject with DCIS, that is HER2 positiveand SIAH2 positive; and administering an aggressive breast cancertherapy to the subject.

In some embodiments, a method of identifying a subject who will not beresponsive to radiation therapy is provided. The method comprisesidentifying a subject with DCIS at an elevated risk of invasive breastcancer; and determining if the subject is HER2 (or EGFR) and SIAH2positive, wherein if the subject is HER2 and SIAH2 positive,administering an aggressive therapy to the subject, wherein theaggressive therapy is not radiation therapy, and wherein the aggressivetherapy is selected from the group consisting of: an antibody to HER2 orTrastuzumab.

In some embodiments, a method of identifying a subject for an aggressivecancer therapy is provided. The method comprises identifying a subjectwith DCIS at an elevated risk of invasive breast cancer; and determiningif the subject is HER2 and SIAH2 positive, wherein if the subject isHER2 and SIAH2 positive, administering an aggressive therapy to thesubject, wherein the aggressive therapy is not radiation therapy, andwherein the aggressive therapy is selected from the group consisting of:an antibody to HER2 or Trastuzumab.

In some embodiments, a method of determining which method of treatmentto recommend to a subject is provided. The method comprises identifyinga subject with DCIS at elevated risk of invasive breast cancer; anddetermining if the subject is HER2 and SIAH2 positive, wherein if thesubject is HER2 and SIAH2 positive, recommending an aggressive therapyto the subject, wherein the aggressive therapy is not radiation therapy,and wherein the aggressive therapy is selected from the group consistingof: an antibody to HER2 or Trastuzumab.

In some embodiments, a method for treating a subject is provided. Themethod comprises providing a DCIS sample from a subject; analyzing theDCIS sample for a level of at least PR, and at least either: a)analyzing the sample for at least HER2 and SIAH2, or b) analyzing thesample for at least FOXA1; and providing a prognosis based upon at leastPR, HER2 and SIAH2 or based upon at least PR and FOXA1, wherein if thesample is PR positive, further analyzing the sample for a level of COX2,wherein COX2 positive with at least FOXA1 positive indicates a high riskof invasive breast cancer, determining if the subject is HER2 positive;and administering an aggressive therapy to the subject if the subject isHER2 positive, wherein the aggressive therapy is not radiation therapy,and wherein the aggressive therapy is selected from the group consistingof: an antibody to HER2 or Trastuzumab.

In some embodiments, a method for decreasing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject; analyzing the DCIS sample for alevel of at least PR, and at least either: analyzing the sample for atleast HER2 and SIAH2, or analyzing the sample for at least FOXA1; andproviding a prognosis based upon at least PR, HER2 and SIAH2 or basedupon at least PR and FOXA1; further analyzing the sample for a level ofKi67, size, or a level of Ki67 and size, if the sample is PR positiveand FOXA1 negative; and wherein if the sample is Ki67 positive, a sizelarger than 5 mm of DCIS, or both, indicates an elevated risk ofinvasive breast cancer; and administering an aggressive therapy to thesubject if the subject is both: HER2 positive, and FOXA1 negative, whenKi67 positive, when a size larger than 5 mm of DCIS, or a combinationthereof, wherein the aggressive therapy is not radiation therapy, andwherein the aggressive therapy is selected from the group consisting of:an antibody to HER2, or Trastuzumab.

In some embodiments, a method of providing a benefit of radiationtherapy is provided. The method comprises: identifying a subject withDCIS at elevated risk of invasive breast cancer; and administeringradiation therapy to the subject if the subject is HER2 negative and notadministering radiation therapy to the subject if the subject tis HER2positive.

In some embodiments, a method for reducing a risk of stage 1A invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject; analyzing the DCIS sample for alevel of at least PR, and at least either: a) analyzing the sample forat least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1;and providing a prognosis based upon at least PR, HER2 and SIAH2 orbased upon at least PR and FOXA1, wherein if the sample is PR positive,further analyzing the sample for a level of COX2, wherein COX2 positivewith at least FOXA1 positive indicates a high risk of invasive breastcancer, and wherein if the risk of the invasive breast cancer is high,providing the subject a more aggressive therapy than standard of care.

In some embodiments, a method of determining if insurance will cover thecost of radiation therapy is provided. The method comprises identifyinga subject at elevated risk of invasive breast cancer and that has DCIS;determining if the subject is HER2 positive; and not covering a cost ofradiation therapy to the subject if the subject is HER2 positive, andcovering the cost of radiation therapy to the subject if the subject isHER2 negative.

In some embodiments, a method of providing reimbursement for a radiationtherapy is provided. The method comprises identifying a subject that hasDCIS and that is further at elevated risk of invasive breast cancer;determining if the subject is HER2 positive and SIAH2 positive; andproviding reimbursement of a cost of radiation therapy to the subject ifthe subject is HER2 negative or SIAH2 negative.

In some embodiments, a method of providing a treatment to a subject whowould not otherwise be treated under a current standard of care isprovided. The method comprises identifying a subject having DCIS,wherein the subject has an elevated risk of developing invasive breastcancer; and administering to the subject chemotherapy, an antibody toHER2, and/or Trastuzumab to the subject if the subject is HER2+ andSIAH+.

In some embodiments, a method of selecting a therapy for a subject isprovided. The method comprises identifying a subject with DCIS at anelevated risk of invasive breast cancer; and

determining if the subject is HER2 positive or HER2 negative, wherein ifthe subject is HER2 positive, administering an aggressive therapy to thesubject, wherein the aggressive therapy is not radiation therapy, andwherein the aggressive therapy is selected from the group consisting of:an antibody to HER2 or Trastuzumab; and wherein if the subject is HER2negative, not administering an aggressive therapy to the subject,thereby reducing that subject's risk of a cardiovascular event.

In some embodiments, a method of treating a subject who will berefractory to radiotherapy is provided. The method comprises identifyinga subject with DCIS, that is HER2 positive and SIAH2 positive; andadministering to the subject a therapy other than radiotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs depicting that almost all elevated riskpatients have a significant benefit from radiation therapy.

FIG. 2 is a set of graphs depicting that there is a subset of people whodo not respond to radiation therapy, who are HER2+ and SIAH2+.

FIG. 3 depicts SIAH2 IHC assays (top, negative, on a UUH TMA; bottom,positive, on a Biomax BR8011 TMA). FIG. 3 are IHC assay images depictingsome embodiments of a negative (top) and positive (bottom) stainingresult for SIAH2.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

While there have been a number of developments in determining the riskthat a subject may have in developing ductal carcinoma in situ (DCIS)and/or invasive breast cancer, the analysis often focuses on stratifyingvarious subjects according to risk alone, rather than on how receptive aparticular subject may be to a particular treatment. The presentdisclosure provides a method for taking the state of the art further,and allows one to properly match a particular subject with a particulartherapy. In some embodiments, by providing a subject having DCIS, who isalso at an elevated risk of invasive breast cancer, one can then testthe subject for the subject's receptiveness to radiation therapy via oneor more marker(s) provided herein. Thus, one can properly pair subjectsat an elevated risk of invasive breast cancer, with a therapy that willwork for the subject. This can be especially effective for determiningwhich subjects should receive radiation therapy, as there are asignificant number of non-responsive subjects who are in the elevatedrisk category for invasive breast cancer. Ideally, such subjects shouldnot receive radiation therapy, but instead an alternative form oftherapy to prevent and/or reduce the risk of the invasive breast cancerevent, such as an anti-HER2 antibody therapy, ERBB therapies, and, forexample ERBB1234. The present disclosure provides a brief set ofdefinitions and embodiments, followed by a detailed description of theprocess and various embodiments around the process, and then concludeswith a number of examples.

Definitions and Optional Embodiments

The term “and/or” shall be taken to provide explicit support for bothmeanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a nucleicacid molecule” includes single or plural nucleic acid molecules and isconsidered equivalent to the phrase “comprising at least one nucleicacid molecule.” The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise. As used herein, “comprises”means “includes.” Thus, “comprising A or B,” means “including A, B, or Aand B,” without excluding additional elements. Unless otherwisespecified, the definitions provided herein control when the presentdefinitions may be different from other possible definitions.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. All HUGO GeneNomenclature Committee (HGNC) identifiers (IDs) mentioned herein areincorporated by reference in their entirety. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

“Antibody” denotes a polypeptide including at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen. In some embodiments, antibodies arecomposed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (VH) region and the variable light(VL) region. Together, the VH region and the VL region are responsiblefor binding the antigen recognized by the antibody. The term antibodyincludes intact immunoglobulins, as well the variants and portionsthereof, such as Fab′ fragments, F(ab)′₂ fragments, single chain Fvproteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFvprotein is a fusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies). See also, Pierce Catalogand Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York, 1997. Variousantibodies can be used for detecting a marker, including for example,those in the following table, Table 0.1.

TABLE 0.1 ANTIBODIES (FOR DETECTION) Antibody Clone (IVD Antibody toListed or ASR), Unless Protein Otherwise Noted Manufacturer(s) PGR (PR)mouse 16 Leica Biosystems and Biocare Medical PGR (PR) mouse PgR 636Dako and Biocare Medical PGR (PR) mouse PgR 1294 Dako PGR (PR) rabbitSP2 Thermo Scientific and Biocare Medical PGR (PR) rabbit SP42 CellMarque PGR (PR) rabbit EP2 BioGenex PGR (PR) rabbit 1E2 Ventana MedicalSystems PGR (PR) mouse PR88 BioGenex PGR (PR) rabbit Y85 Cell MarqueERBB2 (HER2) rabbit SP3 Cell Marque, Thermo Scientific, and DiagnosticBioSystems rabbit polyclonal Dako ERBB2 (HER2) HercepTest/A0485 ERBB2(HER2) mouse CB11 Leica Biosystems, Cell Marque, and Biocare MedicalERBB2 (HER2) rabbit EP3 Cell Marque and BioGenex ERBB2 (HER2) rabbit 4B5Ventana Medical Systems ERBB2 (HER2) rabbit EP1045Y Thermo ScientificMKI67 (Ki-67) mouse MIB-1 Dako and Biocare Medical MKI67 (Ki-67) mouseMM1 Leica Biosystems and Biocare Medical MKI67 (Ki-67) rabbit SP6 CellMarque, Thermo Scientific, Biocare Medical, and Diagnostic BioSystemsMKI67 (Ki-67) mouse K2 Leica Biosystems MKI67 (Ki-67) rabbit 30-9Ventana Medical Systems MKI67 (Ki-67) mouse 7B11 ThermoFisher ScientificMKI67 (Ki-67) rabbit EP5 BioGenex MKI67 (Ki-67) mouse BGX-297 BioGenexMKI67 (Ki-67) mouse Ki88 BioGenex PTGS2 (COX-2) rabbit SP21 Cell Marque,Ventana Medical Systems, Thermo Scientific, Biocare Medical, andDiagnostic BioSystems PTGS2 (COX-2) mouse CX-294 Dako PTGS2 (COX-2)mouse COX 229 ThermoFisher Scientific PTGS2 (COX-2) mouse 4H12Diagnostic BioSystems FOXA1 mouse 2F83 Cell Marque and Ventana MedicalSystems FOXA1 rabbit SP88 (RUO) Spring Bioscience and ThermoFisherScientific FOXA1 rabbit EP277 (RUO) Epitomics INK4A (p16) mouse E6H4Ventana Medical Systems INK4A (p16) mouse G175-405 (RUO) BioGenex and BDPharminGen INK4A (p16) mouse JC8 (RUO) NA INK4A (p16) mouse 6H12 (RUO)NA SIAH2 mouse MRQ-PRE Cell Marque SIAH2 mouse 24E6H3 (RUO) Santa CruzBiotechnology and Novus Biologicals

In some embodiments, any of the methods, kits, etc. provided herein thattest for a presence or absence of any of the target proteins listed intable 0.1, can employ any one or more of the corresponding antibodies tothat target protein.

In some embodiments, each heavy and light chain contains a constantregion and a variable region, (the regions are also known as “domains”).In combination, the heavy and the light chain variable regionsspecifically bind the antigen. Light and heavy chain variable regionscontain a “framework” region interrupted by three hypervariable regions,also called “complementarity-determining regions” or “CDRs.”

References to “VH” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “VL” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “polyclonal antibody” is an antibody that is derived from differentB-cell lines. Polyclonal antibodies are a mixture of immunoglobulinmolecules secreted against a specific antigen, each recognizing adifferent epitope. These antibodies are produced by methods known tothose of skill in the art, for instance, by injection of an antigen intoa suitable mammal (such as a mouse, rabbit or goat) that induces theB-lymphocytes to produce IgG immunoglobulins specific for the antigen,which are then purified from the mammal's serum.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one example, all theCDRs are from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they aresubstantially identical to human immunoglobulin constant regions, e.g.,at least about 85-90%, such as about 95% or more identical. Hence, allparts of a humanized immunoglobulin, except possibly the CDRs, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. Humanized immunoglobulins can be constructedby means of genetic engineering (see for example, U.S. Pat. No.5,585,089).

The term “array” denotes an arrangement of molecules, such as biologicalmacromolecules (such as peptides or nucleic acid molecules) orbiological samples (such as tissue sections), in addressable locationson or in a substrate. A “microarray” is an array that is miniaturized soas to require or be aided by microscopic examination for evaluation oranalysis. Arrays are sometimes called chips or biochips.

The array of molecules makes it possible to carry out a very largenumber of analyses on a sample at one time. In some embodiments, arraysof one or more molecule (such as an oligonucleotide probe) will occur onthe array a plurality of times (such as twice), for instance to provideinternal controls. The number of addressable locations on the array canvary, for example from at least one, to at least 2, to at least 5, to atleast 10, at least 20, at least 30, at least 50, at least 75, at least100, at least 150, at least 200, at least 300, at least 500, least 550,at least 600, at least 800, at least 1000, at least 10,000, or more. Inparticular examples, an array includes nucleic acid molecules, such asoligonucleotide sequences that are at least 15 nucleotides in length,such as about 15-40 nucleotides in length. In particular examples, anarray includes oligonucleotide probes or primers which can be used todetect the markers noted herein, such as at least one of those in Tables1-9, 11 and 13-15 provided herein.

In some embodiments, within an array, each arrayed sample can beaddressable, in that its location can be reliably and consistentlydetermined within at least two dimensions of the array. Addressablearrays can be computer readable, in that a computer can be programmed tocorrelate a particular address on the array with information about thesample at that position (such as hybridization or binding data,including for instance signal intensity). In some examples of computerreadable formats, the individual features in the array are arrangedregularly, for instance in a Cartesian grid pattern, which can becorrelated to address information by a computer.

Protein-based arrays include probe molecules that are or includeproteins, or where the target molecules are or include proteins, andarrays including nucleic acids to which proteins are bound, or viceversa. In some examples, an array contains antibodies to markersprovided herein, such as at least one of those in Tables 1-9, 11 and13-15.

As used herein, the term “gene” means nucleic acid in the genome of asubject capable of being expressed to produce a mRNA and/or protein inaddition to intervening intronic sequences and in addition to regulatoryregions that control the expression of the gene, e.g., a promoter orfragment thereof.

As used herein, the term “diagnosis”, and variants thereof, such as, butnot limited to “diagnose” or “diagnosing” shall include, but not belimited to, a primary diagnosis of a clinical state or any primarydiagnosis of a clinical state. A diagnostic assay described herein isalso useful for assessing the remission of a subject, or monitoringdisease recurrence, or tumor recurrence, such as following surgery,radiation therapy, adjuvant therapy or chemotherapy, or determining theappearance of metastases of a primary tumor.

In some embodiments, a prognostic assay described herein is useful forassessing likelihood of treatment benefit, disease recurrence, tumorrecurrence, or metastasis of a primary tumor, such as following surgery,radiation therapy, adjuvant therapy or chemotherapy. All such uses ofthe assays described herein are encompassed by the present disclosure.In some embodiments, the test can be used to predict if the patient willhave an occurrence.

The term “breast tumor” denotes a neoplastic condition of breast tissuethat can be benign or malignant. The term “tumor” is synonymous with“neoplasm” and “lesion”. Exemplary breast tumors include invasive breastcancer, DCIS, lobular carcinoma in situ (LCIS), and atypical ductalhyperplasia (ADH).

The term “cancer” denotes a malignant neoplasm that has undergonecharacteristic anaplasia with loss of differentiation, increased rate ofgrowth, invasion of surrounding tissue, and is capable of metastasis.The term “cancer” shall be taken to include a disease that ischaracterized by uncontrolled growth of cells within a subject, such as,but not limited to, invasive breast cancer.

The term “intraductal lesion” refers to tumors that are confined to theinterior of the mammary ducts and are, therefore, not invasive breastcancers. Exemplary intraductal lesions include ADH and DCIS.

ADH is a neoplastic intraductal (non-invasive) lesion characterized byproliferation of evenly distributed, monomorphic mammary epithelialcells.

DCIS is a neoplastic intraductal (non-invasive) lesion characterized byincreased mammary epithelial proliferation with subtle to markedcellular atypia. DCIS has been divided into grades (low, intermediate,and high) based on factors such as nuclear atypia, intraluminalnecrosis, mitotic activity etc. Low-grade DCIS and ADH aremorphologically identical, and ADH is distinguished from DCIS based onthe extent of the lesion, as determined by its size and/or the number ofinvolved ducts. DCIS is initially typically diagnosed from a tissuebiopsy triggered by a suspicious finding (e.g., microcalcifications,unusual mass, tissue distortion or asymmetry, etc.) on a mammogramand/or ultrasound imaging test. It may be from routine screening imagingor, more rarely, from diagnostic imaging triggered by a positivephysical examination (e.g., a palpable mass, nipple discharge, skinchange, etc.) or by a significant change in a previously identifiedmass.

Cellular proliferation in DCIS is confined to the milk ducts. If theproliferating cells have invaded through the basement membrane of themyoepithelial cell (MEC) layer lining the duct, thus appearing in thesurrounding stroma, then the lesion is considered an invasive breastcancer, even if DCIS is also present. In some cases, the invasion isvery minimal (microinvasion) or the only evidence of invasion isdisruption of the MEC layer (e.g., by observing discontinuities inMEC-specific protein marker stains such as SMMHC and/or p63). Typically,these microinvasive cases are treated as invasive breast cancers,although there is some controversy in the treatment of these cases.

Recurrence rates in DCIS with current treatments are difficult toestimate. However, it is likely that about 20% of patients who receivelumpectomies without any further treatment would experience recurrenceevents within 10 years, approximately evenly split between DCIS andinvasive events, while <2% of patients who receive mastectomies wouldexperience recurrence. Standard of care with lumpectomy is to receiveadjuvant radiation therapy (RT). Several randomized clinical trialsprovide evidence that adjuvant radiation therapy following lumpectomyreduces recurrence risk by approximately half for both DCIS and invasiveevent types, and that current clinical and pathologic assessmenttechniques cannot identify a low-risk sub-group in which there is nobenefit from radiation therapy. Radiation is not typically administeredafter mastectomy. Importantly, although radiation reduces the risk ofrecurrence events, a survival benefit has not been established withradiation like it has for invasive breast cancer.

LCIS is non-invasive lesion that originates in mammary terminalduct-lobular units generally composed of small and often looselycohesive cells. When it has spread into the ducts, it can bedifferentiated from DCIS based on morphology and/or marker stains.

The term “invasive breast cancer” denotes a malignant tumor distinctfrom, and non-overlapping with, ADH and DCIS, in which the tumor cellshave invaded adjacent tissue outside of the mammary duct structures. Itcan be divided into stages (I, IIA, IIB, IIIA, IIIB, and IV).

Surgery is a treatment for a breast tumor and is frequently involved indiagnosis. The type of surgery depends upon how widespread the tumor iswhen diagnosed (the tumor stage), as well as the type and grade oftumor. The term “treatment” as provided herein does not require thecomplete or 100% curing of the subject. Instead, it encompasses thebroader concept or delaying the onset of one or more symptoms, extendingthe life and/or quality of life of the subject, reducing the severity ofone or more symptoms, etc.

“Risk” herein is the likelihood for a subject diagnosed with DCIS tohave a subsequent ipsilateral breast event after having a first DCISevent. Primary treatment for DCIS can include surgery, radiation, or anadjuvant chemotherapy. In some embodiments, the initial DCIS can beremoved. The event can be a DCIS event or an invasive breast cancerevent. “Risk of invasive breast cancer”, denotes a risk of developing(or being diagnosed with) a subsequent invasive breast cancer in thesame (a.k.a. ipsilateral) breast. That is also true for “risk of DCIS”or total risk. In some embodiments, the initial DCIS can be removed.

In some embodiments, surgery as a treatment for DCIS breast tumorsand/or preventing or reducing the risk of subsequent ipsilateralinvasive breast cancer can include a lumpectomy, mastectomy, and/orbilateral mastectomy.

Adjuvant chemotherapy is often used after surgery to treat any residualdisease. Systemic chemotherapy often includes a platinum derivative witha taxane. Adjuvant chemotherapy is also used to treat subjects who havea recurrence or metastasis.

“Adjuvant DCIS treatment” denotes any treatment that is appropriate fora subject that is likely to have a subsequent DCIS event, which caninclude, less aggressive to more aggressive treatment options dependingon the risk profile and perceived patient benefit, from frequentmonitoring with planned subsequent lumpectomy upon early detection of abreast event, to lumpectomy without radiation, to an additionallumpectomy, to wide excision. In some embodiments, a subject at risk ofDCIS recurrence, but not invasive breast cancer can receive adjuvantDCIS treatment (optionally, in combination with any of the embodimentsprovided herein).

“Adjuvant invasive breast cancer treatment” denotes any treatment thatis appropriate for a subject that is likely to have an invasive breastcancer occurrence, which can include, lumpectomy with radiation, tolumpectomy with a receptor targeted chemotherapy, to lumpectomy withradiation with a receptor targeted chemotherapy, to mastectomy, tomastectomy with a receptor targeted chemotherapy, to mastectomy withradiation, to mastectomy with radiation and a receptor targetedchemotherapy, to surgery with a chemotherapy. In some embodiments, asubject at risk of DCIS recurrence, but not invasive breast cancer canreceive adjuvant DCIS treatment (optionally, in combination with any ofthe embodiments provided herein).

A “marker” refers to a measured biological component such as a protein,mRNA transcript, or a level of DNA amplification. The risk of asubsequent ipsilateral breast event can be predicted through varioussets or markers that in combination allow for the prediction of whetheror not a subject who has DCIS is likely to experience an ipsilateralDCIS recurrence, a subsequent ipsilateral invasive breast cancer, both,or neither following treatment for DCIS.

The term “control” refers to a sample or standard used for comparisonwith a sample which is being examined, processed, characterized,analyzed, etc. In some embodiments, the control is a sample obtainedfrom a healthy patient or a non-tumor tissue sample obtained from apatient diagnosed with a breast tumor. In some embodiments, the controlis a historical control or standard reference value or range of values(such as a previously tested control sample, such as a group of breasttumor patients with poor prognosis, or group of samples that representbaseline or normal values, such as the level of cancer-associated genesin non-tumor tissue).

The “Cox hazard ratio” is derived from the Cox proportional hazardsmodel. Proportional hazards models are a class of survival models instatistics. Survival models relate the time that passes before someevent occurs to one or more covariates that may be associated with thatquantity of time. In the Cox proportional hazards model, the uniqueeffect of a unit increase in a covariate is multiplicative with respectto the hazard rate. A “Cox hazard ratio” is the ratio of the hazardrates corresponding to the conditions described by two levels of anexplanatory variable—a covariate, that is calculated using the coxproportional hazards model. The cox hazard ratio is the ratio ofsurvival hazards for a one-unit change in the covariate. For example,the Cox hazard ratio may be the ratio of survival hazards for a 1 unitchange in the logarithmic gene expression level. Thus, a larger valuehas a greater effect on survival or the hazard rate of the event beingassessed, such as disease recurrence. In some embodiments, a hazardratio (HR) greater than 1 indicates that an increased covariate level isassociated with a worse patient outcome, where the covariate level is amarker expression level. In some embodiments, a HR less than 1 indicatesthat a decreased covariate level is associated with a better patientoutcome, where the covariate level is a marker expression level.

As used herein, the term “non-tumor tissue sample” shall be taken toinclude any sample from or including a normal or healthy cell or tissue,or a data set produced using information from a normal or healthy cellor tissue. For example, the non-tumor sample may be selected from thegroup comprising or consisting of: (i) a sample comprising a non-tumorcell; (ii) a sample from a normal tissue; (iii) a sample from a healthytissue; (iv) an extract of any one of (i) to (iii); (v) a data setcomprising measurements of modified chromatin and/or gene expression fora healthy individual or a population of healthy individuals; (vi) a dataset comprising measurements of modified chromatin and/or gene expressionfor a normal individual or a population of normal individuals; and (vii)a data set comprising measurements of the modified chromatin and/or geneexpression from the subject being tested wherein the measurements aredetermined in a matched sample having normal cells. Preferably, thenon-tumor sample is (i) or (ii) or (v) or (vii).

As used herein, the term “subject” encompasses any animal includinghumans, preferably a mammal. Exemplary subjects include but are notlimited to humans, primates, livestock (e.g. sheep, cows, horses,donkeys, pigs), companion animals (e.g. dogs, cats), laboratory testanimals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wildanimals (e.g. fox, deer). Preferably the mammal is a human or primate.More preferably the mammal is a human.

Detecting expression of a gene product denotes determining of a levelexpression in either a qualitative or quantitative manner can detectnucleic acid molecules or proteins. Exemplary methods include, but arenot limited to: microarray analysis, RT-PCR, Northern blot, Westernblot, next generation sequencing, and mass spectrometry.

The term “diagnosis” denotes the process of identifying a disease by itssigns, symptoms and results of various tests. The conclusion reachedthrough that process is also called “a diagnosis.” Forms of testingcommonly performed include biopsy for the collection of the DCIS. Insome embodiments, a diagnosis includes determining whether a subjectwith DCIS has a good or poor prognosis. In some embodiments, theprognosis can be a high or low likelihood of a subsequent (within thenext 10 years, 15, or 20 years) DCIS event. In some embodiments, theprognosis can be a high or low likelihood of a (within the next 10years, 15, or 20 years) invasive breast cancer event. In someembodiments, the prognosis can be a high or low likelihood of asubsequent (within the next 10 years) DCIS event and a high or lowlikelihood of a (within the next 10 years) invasive breast cancer event.

“Differential or alteration in expression” denotes a difference orchange, such as an increase or decrease, in the amount of RNA, theconversion of mRNA to a protein, level of protein in the system, or anycombination thereof. In some examples, the difference is relative to acontrol or reference value or range of values, such as an amount of geneexpression that is expected in a subject who does not have DCIS and/oran invasive breast cancer or in non-tumor tissue from a subject with abreast tumor. Detecting differential expression can include measuring achange in gene expression or a change in protein levels.

The term “expression” denotes the process by which the coded informationof a gene is converted into an operational, non-operational, orstructural part of a cell, such as the synthesis of an RNA and/orprotein. Gene expression can be influenced by external signals. Forinstance, exposure of a cell to a hormone may stimulate expression of ahormone-induced gene. Different types of cells can respond differentlyto an identical signal. Expression of a gene also can be regulatedanywhere in the pathway from DNA to RNA to protein. Regulation caninclude controls on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization or degradation ofspecific protein molecules after they are produced. In some embodiments,gene expression can be monitored to determine the diagnosis and/orprognosis of a subject with DCIS, such as to determine or to predict asubject's likelihood to develop a subsequent DCIS or invasive breastcancer. In some embodiments, mRNA expression can be monitored todetermine the diagnosis and/or prognosis of a subject with DCIS, such asto determine or to predict a subject's likelihood to develop asubsequent DCIS or invasive breast cancer. In some embodiments, proteinexpression can be monitored to determine the diagnosis and/or prognosisof a subject with DCIS, such as to determine or to predict a subject'slikelihood to develop a subsequent DCIS or invasive breast cancer.

The expression of a nucleic acid molecule in a sample can be alteredrelative to a control sample, such as a normal or non-tumor sample.Alterations in gene expression, such as differential expression, includebut are not limited to: (1) overexpression; (2) underexpression; or (3)suppression of expression. Alterations in the expression of a nucleicacid molecule can be associated with, and in fact cause, a change inexpression of the corresponding protein.

In some embodiments, protein expression can also be altered in somemanner to be different from the expression of the protein in a normal(e.g., non-DCIS) situation. This includes but is not necessarily limitedto: (1) expression of an increased amount of the protein compared to acontrol or standard amount; (2) expression of a decreased amount of theprotein compared to a control or standard amount; (3) alteration of thesubcellular localization or targeting of the protein; (4) alteration ofthe temporally regulated expression of the protein (such that theprotein is expressed when it normally would not be, or alternatively isnot expressed when it normally would be); (5) alteration in stability ofa protein through increased longevity in the time that the proteinremains localized in a cell; and (6) alteration of the localized (suchas organ or tissue specific or subcellular localization) expression ofthe protein (such that the protein is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed), each compared to a control or standard.

Controls or standards for comparison to a sample, for the determinationof differential expression, include samples believed to be normal (inthat they are not altered for the desired characteristic, for example asample from a subject who does not have DCIS or who had DCIS but did notexperience any DCIS and/or invasive breast cancer in the 10 yearsfollowing the DCIS event, as well as laboratory values (e.g., range ofvalues), even though possibly arbitrarily set, keeping in mind that suchvalues can vary from laboratory to laboratory. Laboratory standards andvalues can be set based on a known or determined population value andcan be supplied in the format of a graph or table that permitscomparison of measured, experimentally determined values.

As will be appreciated by one of skill in the art, any of the abovecontrols or standards can be provided for any of the methods (such astreatment, analysis, or prognosis) provided herein, and for any of thecompositions or methods. These can be positive or negative controls orstandards (showing, for example, what a high level or normal level ofexpression or presence of the molecule is). The controls can be matchedfor the relevant molecule type as well (e.g., RNA vs. protein). In someembodiments, the control and/or standard can be for COX-2, Ki-67, p16,PR, SIAH2, FOXA1, and/or HER2. In some embodiments, the control and/orstandard can be for COX-2, Ki-67, p16, ER, SIAH2, FOXA1, and/or HER2. Insome embodiments, any of the PR embodiments provided herein can bereplaced with ER as a marker.

The phrase “gene expression profile” (or signature) denotes adifferential or altered gene expression that can be detected by changesin the detectable amount of gene expression (such as cDNA, mRNA, orprotein) or by changes in the detectable amount of proteins expressed bythose genes. A distinct or identifiable pattern of gene expression, forinstance a pattern of high and low expression of a defined set of genesor gene-indicative nucleic acids such as ESTs. In some examples, as fewas two genes provides a profile, but more genes can be used in aprofile, for example, at least 3, 4, 5, 6, or 7 markers (e.g., genes)can be employed to provide a prognosis in regard to risk of subsequentDCIS and/or risk of subsequent invasive breast cancer. Gene expressionprofiles can include relative as well as absolute expression levels ofspecific genes, and can be viewed in the context of a test samplecompared to a baseline or control sample profile (such as a sample fromthe same tissue type from a subject who does not have a tumor). In someembodiments, a gene expression profile in a subject is read on an array(such as a nucleic acid or protein array). For example, a geneexpression profile can be performed using a commercially available arraysuch as Human Genome GeneChip™ arrays from Affymetrix™ (Santa Clara,Calif.). In some embodiments, any two or more of the markers indicatedin any one of Tables 1-9, 11 and 13-15 can be employed as a profile. Theterm “hybridization” means to form base pairs between complementaryregions of two strands of DNA, RNA, or between DNA and RNA, therebyforming a duplex molecule, for example. Hybridization conditionsresulting in particular degrees of stringency will vary depending uponthe nature of the hybridization method and the composition and length ofthe hybridizing nucleic acid sequences. Generally, the temperature ofhybridization and the ionic strength (such as the sodium concentration)of the hybridization buffer will determine the stringency ofhybridization. Calculations regarding hybridization conditions forattaining particular degrees of stringency are discussed in Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring HarborLaboratory, Plainview, N.Y. (chapters 9 and 11).

The term “isolated” as used in an “isolated” biological component (suchas a nucleic acid molecule, protein, or cell) is one that has beensubstantially separated or purified away from other biologicalcomponents in the cell of the organism, or the organism itself, in whichthe component naturally occurs, such as other chromosomal andextra-chromosomal DNA and RNA, proteins and cells. Nucleic acidmolecules and proteins that have been “isolated” include nucleic acidmolecules and proteins purified by standard purification methods. Theterm also embraces nucleic acid molecules and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acid molecules and proteins. In some embodiments, an isolatedcell is a DCIS cell that is substantially separated from other breastcell types, such as non-tumor breast cells.

The term “label” or “probe” denotes an agent capable of detection, forexample by ELISA, spectrophotometry, flow cytometry, or microscopy. Forexample, a label can be attached to a nucleic acid molecule or protein(such as one that can hybridize or bind to any of the markers in any oneor more of Tables 1-9, 11 and 13-15), thereby permitting detection ofthe nucleic acid molecule or protein. Examples of labels include, butare not limited to, radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent agents, fluorophores, haptens, enzymes, andcombinations thereof. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998). In someembodiments, a label is conjugated to a binding agent that specificallybinds to one or more of the markers disclosed in any one or more ofTables 1-9, 11 and 13-15 to allow for detecting the presence of themarker in a subject or a DCIS sample from the subject.

The term “mammal” includes both human and non-human mammals. Examples ofmammals include, but are not limited to: humans, pigs, cows, goats,cats, dogs, rabbits, rats, and mice.

A nucleic acid array is an arrangement of nucleic acids (such as DNA orRNA) in assigned locations on a matrix, such as that found in cDNAarrays, or oligonucleotide arrays.

A “nucleic acid molecules representing genes” is any nucleic acid, forexample DNA (intron or exon or both), cDNA, or RNA (such as mRNA), ofany length suitable for use as a probe or other indicator molecule, andthat is informative about the corresponding gene, such as those listedin Tables 1-9, 11 and 13-15.

“Polymerase chain reaction” (PCR) is an in vitro amplification techniquethat increases the number of copies of a nucleic acid molecule (forexample, a nucleic acid molecule in a sample or specimen), such asamplification of a nucleic acid molecule listed in Tables 1-9, 11 and13-15. The product of a PCR can be characterized by standard techniquesknown in the art, such as electrophoresis, restriction endonucleasecleavage patterns, oligonucleotide hybridization or ligation, and/ornucleic acid sequencing. In some examples, PCR utilizes primers, forexample, DNA oligonucleotides 10-100 nucleotides in length, such asabout 15, 20, 25, 30 or 50 nucleotides or more in length (such asprimers that can be annealed to a complementary target DNA strand bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, such as those listed in Tables 1-9, 11 and 13-15).Primers can be selected that include at least 15, at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50 ormore consecutive nucleotides of a marker provided herein. Methods forpreparing and using nucleic acid primers are described, for example, inSambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989), Ausubel et al. (ed.) (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1998), and Innis et al. (PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.,San Diego, Calif., 1990).

The term “prognosis” denotes a prediction of the course of a disease. Insome embodiments provided herein, the phrase, when used in the contextof a person already having DCIS, denotes the likelihood that a subjecthaving the DCIS will go on (within a following ten, fifteen, or twentyyear period) to have a subsequent a) ipsilateral DCIS event aftersurgical removal of the primary DCIS, b) ipsilateral invasive breastcancer, c) both events, or d) neither a) nor b). The prediction caninclude determining a) the likelihood of an ipsilateral breast event, b)the likelihood of an ipsilateral breast event in a particular amount oftime (e.g., 1, 2, 3 or 5 years), c) the likelihood that a particulartherapy (e.g., radiation) will prevent an ipsilateral breast event, d)an optimal treatment to help prevent an ipsilateral event that matchesthe severity of the most likely event, or e) combinations thereof.

The phrase “specific binding agent” denotes an agent that bindssubstantially or preferentially only to a defined target such as aprotein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinantvector or a small molecule. In an example, a “specific binding agent” iscapable of binding to at least one of the disclosed markers (such asthose listed in Tables 1-9, 11 and 13-15). In some embodiments, thespecific binding agent is capable of binding to a downstream factorregulated by at least one of the disclosed markers (such as those listedin Tables 1-9, 11 and 13-15). Thus, a nucleic acid-specific bindingagent binds substantially only to the defined nucleic acid, such as RNA,or to a specific region within the nucleic acid. For example, a“specific binding agent” includes an antisense compound (such as anantisense oligonucleotide, siRNA, miRNA, shRNA or ribozyme) that bindssubstantially to a specified RNA.

A “protein-specific binding agent” binds substantially only the definedprotein, or to a specific region within the protein. For example, a“specific binding agent” includes antibodies and other agents that bindsubstantially to a specified polypeptide. Antibodies can be monoclonalor polyclonal antibodies that are specific for the polypeptide, as wellas immunologically effective portions (“fragments”) thereof. Thedetermination that a particular agent binds substantially only to aspecific polypeptide may readily be made by using or adapting routineprocedures. One suitable in vitro assay makes use of the Westernblotting procedure (described in many standard texts, including Harlowand Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Cyclooxygenase-2 (“prostaglandin-endoperoxide synthase 2,” “PTGS2,” and“COX-2”; HGNC:9605), referenced herein as COX-2, is an enzyme that isencoded by the PTGS2 gene. Unless denoted otherwise, the term canencompass DNA, RNA, and/or protein versions. Thus, a level of theindicated marker can denote, for example, RNA levels or protein levels.The use of the generic term herein (such as a “level of COX-2”), denotesall of the above options together and individually (e.g., COX-2 proteinlevel and COX-2 RNA level, or COX-2 protein level, or COX-2 RNA level).

Marker of proliferation Ki-67 (“MKI67” and “MIB-1”; HGNC:7107),referenced herein as Ki-67, is a protein that is encoded by the MKI67gene. Unless denoted otherwise, the term can encompass DNA, RNA, and/orprotein versions. Thus, a level of the indicated marker can denote, forexample, RNA levels or protein levels. The use of the generic termherein (such as a “level of p16”), denotes all of the above optionstogether and individually (e.g., Ki-67 protein level and Ki-67 RNAlevel, or Ki-67 protein level, or Ki-67 RNA level).

p16 isoform of cyclin-dependent kinase inhibitor 2A (“cyclin-dependentkinase inhibitor 2A,” “p16/INK4A,” “CDKN2A,” and “MTS1”; HGNC:1787),referenced herein as “p16”, is a tumor suppressor protein that isencoded by the CDKN2A gene. Unless denoted otherwise, the term canencompass DNA, RNA, and/or protein versions. Thus, a level of theindicated marker can denote, for example, RNA levels or protein levels.The use of the generic term herein (such as a “level of p16”), denotesall of the above options together and individually (e.g., p16 proteinlevel and p16 RNA level, or p16 protein level, or p16 RNA level).

Progesterone receptor (“NR3C3,” “PR,” and “PGR”; HGNC:8910), referencedherein as “PR”, is a protein that is encoded by the PGR gene. Unlessdenoted otherwise, the term can encompass DNA, RNA, and/or proteinversions. Thus, a level of the indicated marker can denote, for example,RNA levels or protein levels. The use of the generic term herein (suchas a “level of PR”), denotes all of the above options together andindividually (e.g., PR protein level and PR RNA level, or PR proteinlevel, or PR RNA level).

Estrogen receptor 1 (“ESR1,” “ER,” “ESR,” “Era,” “ESRA,” “ESTRR,” and“NR3A1”; HGNC:3467), referenced herein as “ER”, is a protein that isencoded by the ESR1 gene. Unless denoted otherwise, the term canencompass DNA, RNA, and/or protein versions. Thus, a level of theindicated marker can denote, for example, RNA levels or protein levels.The use of the generic term herein (such as a “level of ER”), denotesall of the above options together and individually (e.g., ER proteinlevel and ER RNA level, or ER protein level, or ER RNA level).

SIAH2 E3 ubiquitin protein ligase 2 (“SIAH2” and “seven in absentia[Drosophila] homolog 2”; HGNC:10858), referenced herein as SIAH2, is anenzyme that is encoded by the SIAH2 gene. Unless denoted otherwise, theterm can encompass DNA, RNA, and/or protein versions. Thus, a level ofthe indicated marker can denote, for example, RNA levels or proteinlevels. The use of the generic term herein (such as a “level” ofSIAH2”), denotes all of the above options together and individually(e.g., SIAH2 protein level and SIAH2 RNA level, or SIAH2 protein level,or SIAH2 RNA level).

forkhead box A1 (“FOXA1”; HGNC:5021), referenced herein as FOXA1, is aprotein that is encoded by the FOXA1 gene. Unless denoted otherwise, theterm can encompass DNA, RNA, and/or protein versions. Thus, a level ofthe indicated marker can denote, for example, RNA levels or proteinlevels. The use of the generic term herein (such as a “level of FOXA1”),denotes all of the above options together and individually (e.g., FOXA1protein level and FOXA1 RNA level, or FOXA1 protein level, or FOXA1 RNAlevel).

v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2″(“ERBB2,” “HER2” [human epidermal growth factor receptor 2], “NEU”, and“CD340”; HGNC:3430), referenced herein as “HER2”, is a protein that isencoded by the ERBB2 gene. Unless denoted otherwise, the term canencompass DNA, RNA, and/or protein versions. Thus, a level of theindicated marker can denote, for example, RNA levels or protein levels.The use of the generic term herein (such as a “level of HER2”), denotesall of the above options together and individually (e.g., HER2 proteinlevel and HER2 RNA level, or HER2 protein level, or HER2 RNA level).

A subject having “post menopausal” status can be identified by menstrualcessation (if known) or by age (if menstrual status not known) forexample, greater than 50, such as greater than 55.

The term “radiation therapy” denotes a therapy that involves or includessome form of radiation in an amount that is therapeutic to the subject.

The term “non-radiation therapy” denotes a therapy that is adequate foraddressing or reducing the risk of invasive breast cancer in a subject,and that does not derive its therapeutic effect by radiation. Examplesof such therapy include, chemo therapeutics, targeted and non targeted,immune and non-immune modulated, monoclonal, other targeted andnon-targeted, genomic therapies, antibody therapeutics, including, HER2antibodies, including Trastuzumab. Often, in the present application,“non-radiation therapy” is denoted as “other therapy”.

The term “aggressive” as used herein denotes that treatment isappropriate for a subject who is at a high risk of developing thedenoted event. Thus, an aggressive breast cancer therapy is a therapyfor a subject who, it is understood, will most likely develop breastcancer. Such therapies are generally more extensive in nature than othertherapies. Examples of such therapies include: aggressive radiationtherapy, and aggressive non-radiation therapy.

General Description of Various Embodiments:

Provided herein are methods for identifying and treating varioussubjects with an appropriate form of therapy, both for the risk to thesubject and for the likelihood that the subject will be responsive tothe therapy. It has been appreciated that not all subjects, even thoseat elevated risk of invasive breast cancer, will respond to variousforms of therapy, and radiation therapy in particular. Thus, variousembodiments provided herein allow one to determine if the subject atelevated risk of invasive breast cancer should receive radiation therapyor some other therapy instead.

In some embodiments, a method of treating a subject is provided. Themethod comprises identifying a subject with DCIS. The subject also hasan elevated level of activity in a k-ras pathway. The subject is thentreated with (or receives) an aggressive breast cancer therapy. In someembodiments, the k-ras pathway is elevated if there is an elevated levelof at least one of: K-ras, RAF, MAPK, MEK, ETS or SIAH2. In someembodiments, elevated denotes at least 10% or more of an increase in theprotein or RNA levels of one or more of K-ras, RAF, MAPK, MEK, ETS orSIAH2. In some embodiments, it is at least 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500% or more than the level in a subject who isnot at elevated risk of developing invasive breast cancer. In someembodiments, the subject is further treated with a non-radiation,aggressive, therapy, if there is an elevated level of at least one ofthe following: K-ras, RAF, MAPK, MEK, ETS or SIAH. In some embodiments,the subject also has DCIS. In some embodiments, the subject has DCIS, isHER2 positive and has elevated levels in 2, 3, 4, 5, or all 6 of: K-ras,RAF, MAPK, MEK, ETS, and SIAH. In some embodiments, the subject has DCISand is HER2 positive and has elevated levels in 1, 2, 3, 4, 5, or all 6of: K-ras, RAF, MAPK, MEK, ETS, and SIAH2, and is then treated with anon-radiation therapy, for example a HER2 antibody, such as trastuzumab.

In some embodiments, a method of treating a subject is provided. Themethod comprises identifying a subject with DCIS, that is HER2 positiveand SIAH2 positive and administering an aggressive breast cancer therapyto the subject. In some embodiments, the treatment is not a radiationtherapy. In some embodiments, the aggressive breast cancer therapy ischemotherapy, such as a Her2 Ab, such as trastuszumab.

In some embodiments, a method of identifying a subject who will not beresponsive to radiation therapy is provided. The method comprises:identifying a subject with DCIS at an elevated risk of invasive breastcancer, and determining if the subject is HER2 (or EGFR) and SIAH2positive. If the subject is HER2 and SIAH2 positive, one thenadministers an aggressive therapy to the subject (or if one is thepatient, one receives the aggressive therapy). The aggressive therapy isnot radiation therapy, and can be selected from one or more of the groupconsisting of: an antibody to HER2 or Trastuzumab.

In some embodiments, a method of identifying a subject for an aggressivecancer therapy is provided. The method comprises identifying a subjectwith DCIS at an elevated risk of invasive breast cancer and determiningif the subject is HER2 and SIAH2 positive. In some embodiments, if thesubject is HER2 and SIAH2 positive, one administers (or instructs theadministration of) an aggressive therapy to the subject. The aggressivetherapy is not radiation therapy. In some embodiments, the aggressivetherapy is selected from one or more of the group consisting of: anantibody to HER2, Trastuzumab, cytotoxic drugs, and ERBB2 directedcompounds (such as antibodies to ERBB2).

In some embodiments, a method for treating a subject is provided. Themethod comprises providing a DCIS sample from a subject, analyzing theDCIS sample for a level of at least PR, and at least either: a)analyzing the sample for at least HER2 and SIAH2, or b) analyzing thesample for at least FOXA1, and providing a prognosis based upon at leastPR, HER2 and SIAH2 or based upon at least PR and FOXA1. If the sample isPR positive, further analyzing the sample for a level of COX2, whereinCOX2 positive with at least FOXA1 positive indicates a high risk ofinvasive breast cancer. The method further comprises determining if thesubject is HER2 positive, and administering an aggressive therapy to thesubject if the subject is HER2 positive. The aggressive therapy is notradiation therapy. In some embodiments, the aggressive therapy isselected from one or more of the group consisting of: an antibody toHER2 and Trastuzumab.

In some embodiments, a method for decreasing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the DCIS sample for alevel of at least PR, and at least either: a) analyzing the sample forat least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1;and then providing a prognosis based upon at least PR, HER2 and SIAH2 orbased upon at least PR and FOXA1. One then further analyzes the samplefor a level of Ki67, size, or a level of Ki67 and size, if the sample isPR positive and FOXA1 negative. If the sample is Ki67 positive, a sizelarger than 5 mm of DCIS, or both, it indicates an elevated risk ofinvasive breast cancer. The method further comprises administering anaggressive therapy to the subject if the subject is both: a) HER2positive, and b) FOXA1 negative, when Ki67 positive, when a size largerthan 5 mm of DCIS, or a combination thereof. The aggressive therapy isnot radiation therapy. In some embodiments, the aggressive therapy isselected from one or more of the group consisting of: an antibody toHER2 and Trastuzumab.

In some embodiments, a method of providing a benefit of radiationtherapy to a subject is provided. The method comprises identifying asubject with DCIS at elevated risk of invasive breast cancer andadministering radiation therapy to the subject if the subject is HER2negative. In some embodiments, one does not administer radiation therapyto the subject if the subject tis HER2 positive.

In some embodiments, a method of determining which method of treatmentto recommend to a subject is provided. The method comprises identifyinga subject with DCIS at elevated risk of invasive breast cancer anddetermining if the subject is HER2 and SIAH2 positive. If the subject isHER2 and SIAH2 positive, one recommends an aggressive therapy to thesubject, wherein the aggressive therapy is not radiation therapy. Insome embodiments, the aggressive therapy for breast cancer is selectedfrom the group consisting of: an antibody to HER2 or Trastuzumab or anyof the other options noted herein. If the subject is HER2 or SIAH2negative, and still at elevated risk of invasive breast cancer, onerecommends radiation therapy.

In some embodiments, recommending is done by a physician to the subject.In some embodiments, this is done separately, following an analysis ofthe markers, by a healthcare provider or via an insurance company. Insome embodiments, the recommending process is provided via the selectionand/or administration of the particular therapy to the subject. In someembodiments, the recommending process is done via the approval ofreimbursement and/or payment of a non-radiation therapy for the subject.

In some embodiments, a method of selecting a therapy for a subject isprovided. The method comprises identifying a subject with DCIS at anelevated risk of invasive breast cancer and determining if the subjectis HER2 positive or HER2 negative. If the subject is HER2 positive, onecan then administer an aggressive therapy to the subject. In someembodiments, the aggressive therapy is not radiation therapy. In someembodiments, the aggressive therapy is selected from the groupconsisting of at least one of: an antibody to HER2 or Trastuzumab orother options disclosed herein. If the subject is HER2 negative, onedoes not administer an aggressive therapy to the subject. Thiscombination allows one to reduce that subject's risk of a cardiovascularevent, while retaining a benefit of treatment for those in need and thatwill benefit from the particular therapy.

In some embodiments, a method of providing a treatment to a subject whowould not otherwise be treated under a current (2018, in the UnitedStates) standard of care is provided. The method comprises identifying asubject having DCIS. The subject also has an elevated risk of developinginvasive breast cancer. The method further comprises administering tothe subject chemotherapy. The chemotherapy can include, for example, anantibody to HER2, and/or Trastuzumab. This is done if the subject isHER2+ and SIAH+.

In some embodiments, a method of treating a subject who will berefractory to radiotherapy is provided. The method comprises identifyinga subject that has DCIS, that is HER2 positive and SIAH2 positive andadministering to the subject a therapy other than radiotherapy. In someembodiments, the therapy other than radiotherapy is an antibody to HER2and/or trastuzumab and/or cytotoxic drugs, and ERBB2 directed compounds(such as antibodies to ERBB2).

In some embodiments, a method for reducing a risk of a stage 1A invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the DCIS sample for alevel of at least PR, and at least either: a) analyzing the sample forat least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1.The method further comprises providing a prognosis based upon at leastPR, HER2 and SIAH2 or based upon at least PR and FOXA1. If the sample isPR positive, further analyzing the sample for a level of COX2. If thesample is COX2 positive with at least FOXA1 positive, it indicates ahigh risk of invasive breast cancer. If the risk of the invasive breastcancer is high, providing the subject a more aggressive therapy than thestandard of care for treating DCIS as of 2018 in the United States(e.g., 1A DCIS only).

In some embodiments, a method of determining if insurance will cover thecost of radiation therapy is provided. The method comprises identifyinga subject at elevated risk of invasive breast cancer and that has DCIS,determining if the subject is HER2 positive, and not covering a cost ofradiation therapy to the subject if the subject is HER2 positive, andcovering the cost of radiation therapy to the subject if the subject isHER2 negative.

In some embodiments, a method of determining if insurance will cover thecost of radiation therapy is provided. The method comprises identifyinga subject at elevated risk of invasive breast cancer and that has DCIS,determining if the subject is HER2 positive and SIAH2 positive, and notcovering a cost of radiation therapy to the subject if the subject isHER2 positive and SIAH2 positive, and covering the cost of radiationtherapy to the subject if the subject is HER2 negative and/or SIAH2negative.

In some embodiments, a method of paying for radiation therapy isprovided. The method comprises identifying a subject at elevated risk ofinvasive breast cancer and that has DCIS, determining if the subject isHER2 positive, and not paying for radiation therapy for the subject ifthe subject is HER2 positive, and paying (at least in part) forradiation therapy for the subject if the subject is HER2 negative.

In some embodiments, a method of paying for radiation therapy isprovided. The method comprises identifying a subject at elevated risk ofinvasive breast cancer and that has DCIS, determining if the subject isHER2 positive and SIAH2 positive, and not paying for radiation therapyfor the subject if the subject is HER2 positive and SIAH2 positive, andpaying (at least in part) for radiation therapy for the subject if thesubject is HER2 negative and/or SIAH2 negative.

In some embodiments, a method of providing reimbursement for a radiationtherapy is provided. The method comprises identifying a subject that hasDCIS and that is further at elevated risk of invasive breast cancer,determining if the subject is HER2 positive and SIAH2 positive, andproviding reimbursement of a cost of radiation therapy to the subject ifthe subject is HER2 negative or SIAH2 negative.

In some embodiments, a method of providing reimbursement fornon-radiation therapy is provided. The method comprises identifying asubject that has DCIS and that is further at elevated risk of invasivebreast cancer, determining if the subject is HER2 positive and SIAH2positive, and providing reimbursement of a cost of non-radiation therapyto the subject if the subject is HER2 positive and SIAH2 positive.

In some embodiments, the aggressive therapy employed herein is a therapythat is appropriate for a subject who is at an elevated risk ofdeveloping invasive breast cancer, and the therapy is adequate toaddress and meaningfully reduce the risk of invasive breast cancer. Insome embodiments, the therapy is a chemotherapy. In some embodiments,the chemotherapy is selected from the group consisting of: an antibodyto HER2 and/or trastuzumab and/or cytotoxic drugs, and ERBB2 directedcompounds (such as antibodies to ERBB2). In some embodiments, thetherapy is a therapy that comprises an anti-HER2 antibody, such as, forexample: trastuzumab. In some embodiments, the therapy comprisestrastuzumab.

In some embodiments, the analysis of each marker is carried out inparallel with each other. In some embodiments, the analysis of eachmarker is carried out at overlapping times. In some embodiments, PRanalysis occurs first and any further analysis depends upon the resultof the PR analysis. In some embodiments, no additional markers arelooked at to determine the particular therapy to administer. In someembodiments, the HER2 and/or SIAH2 analysis is done first. In someembodiments, the sample and/or subject is first identified as havingDCIS, and only after that, is it determined if they have an elevatedrisk of invasive cancer, and only after that, is it determined if theywill be refractory to radiation therapy (i.e., HER2+ and SIAH+).

In some embodiments, additional factors that can be reviewed todetermine the appropriate therapy are those that indicate an elevationin the K-ras pathway. Subjects with elevated levels can be identified asrefractory for radiation therapy (if they also have DCIS).

Exemplary Embodiments Regarding “Elevated Risk” Subjects and theIdentification Thereof

Provided below are exemplary embodiments for identifying subjects atelevated risk of developing invasive breast cancer. While this is notexhaustive, it is representative of how those of skill in the art canidentify such individuals. In some embodiments, any of the methodsprovided herein for identifying a subject at “elevated risk,” or “highrisk” or that should receive a therapy that is “aggressive” indicatesthat the subject is at elevated risk of invasive breast cancer and wouldbenefit from the arrangement provided herein regarding subjects withDCIS and the decision of whether they are refractory to radiationtherapy or not. Thus, any of the embodiments provided herein (regardingtreatment, identification, etc. of subjects at risk of invasive breastcancer, can be used in the embodiments to determine which therapy toadminister to the subject. In some embodiments, any subject that isidentified as having an elevated risk can also be screened for if theywill be responsive to radiation therapy or should instead receive anon-radiation therapy (such as a HER2 antibody). For the prognosislisted in the tables below, only those that indicate an “invasive risk”are relevant for the process provided herein, and only in those thatalready have DCIS. In some embodiments, other categories of subjectsindicate those subjects who would not benefit from the radiationrefractory analysis provided herein. Thus, the disclosure providesguidance as to who would, and who would not, benefit from the presentanalysis. That is, once the elevated risk is established, then furtherexamining the sample to determine the appropriate treatment for thesubject, by the options noted herein (e.g., examining HER2 and SIAH2).

Higher Risk Lower Risk Hazard Row Comment Prognosis Pop. factors factorsRatio P-value n Significance Evaluation (95% # of of Risk Confid.Patients Factors Interval) w/in the population subset indicated Table 11 PR− is an invasive Invasive Age <50 PR− PR+ 6.0 (2.0- 0.00028 168Strong risk factor in risk 18.1) patients younger than 50 2 Thecombination of Invasive Total PR− & PR+ or 4.1 (2.2- 0.000036 603 StrongPR− and Age <50 is risk Age <50 Age ≥50 7.4) an invasive risk factor 3Age alone is a low- Insufficient Total Age <50 Age ≥50 1.8 (1.0- 0.042603 Weak moderate risk for invasive 3.3) factor for invasive prognosisrisk 4 PR status alone is Insufficient Total PR− PR+ 1.6 (0.9- 0.11 603None not sufficient to for invasive 2.9) predict invasive risk prognosisTable 2 1 Elevated SIAH2 is DCIS risk Total HER2+ & HER2− & 2.8 (1.3-0.015 461 Significant a DCIS risk factor SIAH ≥30 SIAH2 <30 6.1) inHER2+ patients 2 Elevated SIAH2 is DCIS risk Total HER2+ & HER2− & 2.2(1.1- 0.04 461 Weak a DCIS risk factor SIAH ≥20 SIAH2 <20 4.6) in HER2+patients 3 HER2 status alone Insufficient Total HER2+ HER2− 1.8 (0.9-0.099 461 None is not sufficient to for DCIS 3.7) predict risk prognosis4 Elevated SIAH2 Insufficient Total SIAH2 ≥30 SIAH2 <30 1.9 (1.0- 0.062461 None alone is not for DCIS 3.9) sufficient to predict prognosis DCISrisk Table 3 1 Elevated SIAH2 is DCIS risk PR− SIAH2 ≥30; SIAH2 <30; 4.1(1.3- 0.011 196 Strong a DCIS risk factor PR− PR− 13.1) in PR− patients2 Elevated SIAH2 is DCIS risk PR− SIAH2 ≥20; SIAH2 <20; 9.2 (1.2- 0.0037196 Strong a DCIS risk factor PR− PR− 70.2) in PR− patients 3 PR statusalone is Insufficient Total PR− PR+ 1.0 (0.5- 0.94 461 None notsufficient to for DCIS 2.0) predict DCIS risk prognosis 4 Elevated SIAH2Insufficient Total SIAH2 ≥20 SIAH2 <20 1.6 (0.8- 0.18 461 None alone isnot for DCIS 3.2) sufficient to predict prognosis DCIS risk Table 4 1Elevated SIAH2 is DCIS risk PR− or SIAH2 ≥30; SIAH2 <30; 3.5 (1.0- 0.026220 Significant a DCIS risk factor HER2+ (PR− or (PR− or 12.1) in PR− orHER2+ HER2+) HER2+) patients 2 Elevated SIAH2 is DCIS risk PR− or SIAH2≥20; SIAH2 <20; 3.3 (1.2- 0.013 220 Significant a DCIS risk factor HER2+(PR− or (PR− or 8.9) in PR− or HER2+ HER2+) HER2+) patients 3 PR− orHER2+ Insufficient Total PR− or PR+ and 1.3 (0.6- 0.49 457 None alone isnot for DCIS HER2+ HER2− 2.5) sufficient to predict prognosis DCIS riskTable 5 1 Elevated SIAH2 is DCIS risk Age >55 SIAH2 ≥30; SIAH2 <30; 4.4(1.8- 0.0014 258 Strong a DCIS risk factor AGE >55 AGE >55 11.0) inpatients older than 55 2 Elevated SIAH2 is DCIS risk Age >55 SIAH2 ≥40;SIAH2 <40; 4.0 (1.6- 0.0054 258 Strong a DCIS risk factor AGE > 55AGE >55 9.9) in patients older than 55 3 Age alone not InsufficientTotal Age >55 Age ≤55 1.1 (0.5- 0.85 461 None sufficient to predict forDCIS 2.1) DCIS risk prognosis 4 Elevated SIAH2 Insufficient Total SIAH2≥30 SIAH2 <30 1.9 (1.0- 0.063 461 None alone is not for DCIS 3.9)sufficient to predict prognosis DCIS risk 5 Elevated SIAH2 InsufficientTotal SIAH2 ≥20 SIAH2 <20 1.6 (0.8- 0.18 461 None alone is not for DCIS3.2) sufficient to predict prognosis DCIS risk 6 Elevated SIAH2Insufficient Total SIAH2 ≥40 SIAH2 <40 1.9 (0.9- 0.10 461 None alone isnot for DCIS 4.0) sufficient to predict prognosis DCIS risk Table 6 1Low FOXA1 is a DCIS risk PR+ FOXA1 ≤150; FOXA1 >150; 2.2 (1.0- 0.049 301Weak DCIS risk factor PR+ PR+ 4.8) within PR+ patients 2 Low FOXA1 is aDCIS risk PR+ FOXA1 ≤100; FOXA1 >100; 2.7 (1.2- 0.018 301 SignificantDCIS risk factor PR+ PR+ 5.8) within PR− patients 3 PR status alone isInsufficient Total PR+ PR− 1.1 (0.6- 0.68 518 None not sufficient to forDCIS 2.1) predict DCIS risk prognosis 4 Low FOXA1 alone InsufficientTotal FOXA1 ≤100 FOXA1 >100 1.6 (0.8- 0.16 518 None is not sufficient tofor DCIS 2.9) predict DCIS risk prognosis 5 Low FOXA1 alone InsufficientTotal FOXA1 ≤150 FOXA1 >150 1.5 (0.8- 0.18 518 None is not sufficient tofor DCIS 2.8) predict DCIS risk prognosis Table 7 1 Low FOXA1 DCIS riskPR+ & FOXA1 ≤150; FOXA1 >150; 3.6 (1.4- 0.0091 175 Strong defined as≤150 is Age <60 PR+ and PR+ and 9.6) a DCIS risk factor AGE <60 AGE <60in PR+ patients younger than 60 2 Low FOXA1 DCIS risk PR+ & FOXA1 ≤100;FOXA1 >100; 3.9 (1.5- 0.0064 175 Strong defined as ≤150 is Age <60 PR+and PR+ and 10.2) a DCIS risk factor AGE <60 AGE <60 in PR+ patientsyounger than 60 3 Low FOXA1 status Insufficient Age <60 FOXA1 ≤100;FOXA1 >100; 2.1 (1.0- 0.065 300 None defined as ≤100 is for DCIS AGE <60AGE <60 4.4) not sufficient to prognosis predict DCIS risk in patientsyounger than 60 4 PR status alone is Insufficient Age <60 PR+; PR+; 1.2(0.6- 0.62 300 None not sufficient to for DCIS AGE <60 AGE <60 2.7)predict DCIS risk in prognosis patients younger than 60 Table 8 1Elevated SIAH2 DCIS risk PR− & SIAH2 ≥30; SIAH2 <30; 6.5 (1.4- 0.0069122 Strong defined as ≥30 is a FOXA1 >100 PR− & PR− & 30.6) DCIS riskfactor in FOXA1 >100 FOXA1 >100 PR− patients with elevated FOXA1 definedas >100 2 The combination of DCIS risk FOXA1 > 100 PR− & (PR+ or 3.9(1.6- 0.0055 328 Strong PR− and elevated SIAH2 ≥30; SIAH2 <30); 9.6)SIAH2 defined as FOXA1 >100 FOXA1 >100 ≥30 is a DCIS risk factor in theelevated FOXA1 category Table 9 1 Combined SIAH2, DCIS risk TOTAL(FOXA1−, OTHER = 4.4 (2.1- 9.09E−06 497 Very Strong FOXA1 PR HER2 PR+)or (FOXA1+ 9.0) and KI67 and ((PR− or PR−) margin status form |HER2+),AND a significant risk SIAH2+) or [PR+ AND prognosis for DCIS((PR+|HER2+), HER2−) KI67+) or |SIAH2−] margin AND positive [(PR− ANDHER2−) |KI67−) AND MARGIN STATUS NEGATIVE 2 Combined SIAH2, DCIS riskTOTAL (FOXA1+. OTHER = 3.0 (1.6- 5.50E−04 497 Strong FOXA1 PR HER2 PR+)or (FOXA1 5.7) and KI67 form a ((PR− or PR−) significant risk |HER2+),AND prognosis for DCIS SIAH2+) or [(PR+ AND ((PR+|HER2+), HER2−)] KI67+)SIAH2- AND [(PR− AND HER2−)] KI67−) 3 Combined SIAH2, DCIS risk TOTAL(FOXA1−, OTHER = 2.6 (1.4- 2.70E−03 497 Significant FOXA1 PR HER2 PR+)or (FOXA1+ 4.9) form a significant ((PR− or PR−) risk prognosis for|HER2+), AND [(PR+ DCIS SIAH2+) AND HER2−) or SIAH2−] 4 Combined SIAH2,DCIS risk RT− (FOXA1−, OTHER = 4.4 (1.9- 2.70E−04 497 Very Strong FOXA1PR HER2 PR+) or (FOXA1+ 10) form a significant ((PR− or PR−) riskprognosis for |HER2+), AND [(PR+ DCIS in RT− SIAH2+); AND RT− HER2−) orSIAH2−] AND RT−

Table 9 provides a summary of the marker combinations that indicate ahigh risk (as used in this table only) of a subject diagnosed with DCISexperiencing an ipsilateral DCIS event after surgical removal of theprimary DCIS.

In some embodiments, Table 11 provides a summary of the combinationsthat indicate a high risk of a subject diagnosed with DCIS experiencingan ipsilateral DCIS and/or invasive breast cancer after surgical removalof the primary DCIS.

TABLE 11 High Risk of Invasive High Risk of DCIS ipsilateral Combinationof Markers ipsilateral event event PR+, FOXA1+ INVASIVE (PR+, FOXA1+) or(KI67+, SIZE >5) INVASIVE PR+ FOXA1+ COX-2+, KI67+ INVASIVE (PR+ FOXA1+COX-2 INVASIVE PR−, HER2−, and SIAH2− INVASIVE PR−, HER2−, and SIAH2−,premenopausal INVASIVE (PR−, HER2−, and SIAH2−) or (PR−, P16 2+) or (PR−P16+KI67+) INVASIVE PR−, FOXA1− INVASIVE PR−, FOXA1− HER2−- INVASIVEPR-, FOXA1-, Pre-menopausal INVASIVE (PR−, FOXA1−, and HER2−) or (PR−,P16 2+) or (PR− P16+ KI67+) INVASIVE (PR−, FOXA1−, and HER2−) or (PR−,HER2−, and SIAH2−) or (PR−, P16 KI67+) INVASIVE (PR+ FOXA1+ COX-2 or(PR-, HER2−, and SIAH2−) INVASIVE (PR+, FOXA1+) or (PR-, HER2-, andSIAH2-) INVASIVE SIAH2 + and PR− DCIS SIAH2 + and FOXA1+ DCIS SIAH2 +and HER2+ DCIS SIAH2 + and post-menopausal DCIS PR+ and FOXA1 − DCISSIAH2 + PR− FOXA1+ DCIS PR− FOXA1+ DCIS PR+and FOXA1 − premenopausalDCIS PR+and FOXA1 − or (KI67+ HER2−) or (KI67+ PR+) DCIS (SIAH2+, PR−FOXA1+) or (KI67+ HER2−) or (KI67+PR+) DCIS (SIAH2+, PR-) or( SIAH2+HER2+) or (KI67+ HER2−) or (KI67+PR+) or (PR+and FOXA1 −) DCIS (SIAH2+,PR− FOXA1 +) or (SIAH2+HER2+) or (KI67+ HER2−) or (KI67+PR+) or (PR+andFOXA1−) DCIS SIAH2 +and PR− postmenopausal DCIS

Table 11 outlines the relevant markers for identifying the level of riskthat a subject with DCIS has for experiencing invasive breast cancer.Table 11 provides a summary of the combinations that indicate a highrisk to a subject who already has DCIS experiencing invasive breastcancer and/or a recurrence of DCIS. The above results (Tables 1-9, 11,13-15) are expressly contemplated for all embodiments of the variousmethods provided herein, as well as kits and compositions, etc.) Asnoted above, in some embodiments, only those who meet the elevated riskof invasive breast cancer are analyzed for HER2 and SIAH2 levels.

In some embodiments, any noted combination of the above markers orvariables can be used for compositions or methods relating to DCISrecurrence and/or a risk of invasive breast cancer (as indicated). Asnoted herein, various combinations denote that the subject is at arelatively higher (or lower) risk of experiencing DCIS recurrence and/ora risk of invasive breast cancer. Thus, in some embodiments, this can bepractically employed in terms of, for example, proper prognosis for thesubject, advanced methods of treatment for the subject (for example,taking an approach that not only resolves DCIS that the subjectcurrently has, but also addresses the risk level of DCIS recurrenceand/or invasive breast cancer appropriately), methods for analyzing asample (for example, a DCIS sample for various markers), compositionsand kits that allow for the above noted methods, etc. In someembodiments, in a PR-background, low FOXA1 correlates with invasiveevents, while in a PR+ background, high FOXA1 correlates with invasiverecurrence. Both correlations are reversed for DCIS events. In someembodiments, separate biomarker-based risk models (algorithms) can beused to predict invasive and DCIS events, and at least some biomarkerscan be assessed differently in the context of other markers, rather thanbeing assigned a single weighting in a linear algorithm. In someembodiments, the PR/FOXA1 combination identifies a subset of patientswho experience a remarkable benefit from RT relative to the remainingpatients. As shown, the studies indicate that the present approach torisk stratification modeling can accurately identify patients at riskfor DCIS or invasive events after a primary DCIS diagnosis. In someembodiments, the models presented here (such as in the Examples below)are the basis of a comprehensive multi-marker panel. It should be notedthat in certain embodiments, the risk models can be performed on acomputing device while in other embodiments, the risk models can beperformed manually.

In some embodiments, a method of analyzing a sample is provided. Themethod comprises analyzing a human DCIS tissue sample for PR, and eitheror both of: analyzing the sample for at least HER2 and SIAH2, and/oranalyzing the sample for at least FOXA1. In some embodiments, dependingupon the nature of the results, this indicates that the subject thatprovided the sample is at a high or elevated risk of invasive breastcancer (see, e.g., Tables 1-9, 11, 13, and 15).

In some embodiments, a method of analyzing a sample is provided. Themethod comprises analyzing a human DCIS tissue sample for a level of atleast SIAH2 and FOXA1. In some embodiments, depending upon the nature ofthe results, this indicates that the subject that provided the sample isat a high or elevated risk of invasive breast cancer (see, e.g., Tables1-9, 11, 13, and 15).

In some embodiments, a method of analyzing a sample is provided. Themethod comprises providing a DCIS sample from a subject having DCIS; 1)analyzing the DCIS sample for SIAH2, and analyzing the DCIS sample forat least one of HER2, PR, FOXA1, or any combination thereof; or 2)analyzing the DCIS sample for FOXA1 and PR. In some embodiments,depending upon the nature of the results, this indicates that thesubject that provided the sample is at a high or elevated risk ofinvasive breast cancer (see, e.g., Tables 1-9, 11, 13, and 15).

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the DCIS sample for alevel of at least PR, and at least either analyzing the sample for atleast HER2 and SIAH2, or analyzing the sample for at least FOXA1. Themethod further comprises providing a prognosis based upon at least PR,HER2 and SIAH2 or based upon at least PR and FOXA1. In some embodiments,depending upon the nature of the results, this indicates that thesubject that provided the sample is at a high or elevated risk ofinvasive breast cancer (see, e.g., Tables 1-9, 11).

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the sample for a levelof at least SIAH2 and FOXA1, and prognosing the subject as having anelevated risk of an invasive breast cancer based upon the level of atleast SIAH2 and FOXA1. In some embodiments, depending upon the nature ofthe results, this indicates that the subject that provided the sample isat a high or elevated risk of invasive breast cancer (see, e.g., Tables1-9, 11).

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the sample for: a) PR,HER2, and SIAH2, or b) PR and FOXA1; and prognosing the subject ashaving an elevated risk of an invasive breast cancer event when at leastone of: a) PR−, HER2−, and SIAH2−, b) PR+, FOXA1+, or c) PR+, FOXA1−,and Ki67+.

In some embodiments, a method for treating a subject at risk of havingan invasive breast cancer is provided. The method comprises providing asubject having DCIS, wherein the subject has a DCIS that is at least oneof: a) PR−, HER2−, and SIAH2−, b) PR+, FOXA1+, or c) PR+, FOXA1−, andKi67+; and administering to the subject a therapy that is moreaggressive than standard of care for DCIS.

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method can involveapplying an algorithm to the protein and/or mRNA expression level as anadditional transformative process, to thereby provide a signature forthe marker. The method comprises providing a DCIS sample from a subject,analyzing the DCIS sample for a level of at least PR, and at leasteither analyzing the sample for at least HER2 and SIAH2, or analyzingthe sample for at least FOXA1. The method further comprises providing aprognosis based upon at least a PR, HER2 and SIAH2 signature or basedupon at least a PR and FOXA1 signature. In some embodiments, dependingupon the nature of the results, this indicates that the subject thatprovided the sample is at a high or elevated risk of invasive breastcancer (see, e.g., Tables 1-9, 11, 13, and 15).

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the sample for a levelof at least SIAH2 and FOXA1, and prognosing the subject as having anelevated risk of an invasive breast cancer based upon the signature ofat least SIAH2 and FOXA1. In some embodiments, depending upon the natureof the results, this indicates that the subject that provided the sampleis at a high or elevated risk of invasive breast cancer (see, e.g.,Tables 1-9, 11, 13, and 15).

In some embodiments, a method for prognosing a risk of an invasivebreast cancer event in a subject is provided. The method comprisesproviding a DCIS sample from a subject, analyzing the sample for: a) PR,HER2, and SIAH2, or b) PR and FOXA1; and prognosing the subject ashaving an elevated risk of an invasive breast cancer event using asignature comprising at least one of: a) PR−, HER2−, and SIAH2−, b) PR+,FOXA1+, or c) PR+, FOXA1−, and Ki67+. In some embodiments, dependingupon the nature of the results, this indicates that the subject thatprovided the sample is at a high or elevated risk of invasive breastcancer (see, e.g., Tables 1-9, 11, 13, and 15).

In some embodiments, any of the methods, compositions, kits, systems,etc. described herein, can be used employing one or more of thefollowing markers and/or combinations (and/or other noted variable),outlined in Tables 1-9 11, 14, and 15 for markers that are relevant toDCIS (Table 9 for the noted combinations) and Tables 11, 13 and 15 formarkers that are relevant to invasive breast cancer. The HER2 and SIAH2analysis processes noted above to determine the appropriate therapy toadminister can be part of or combined with any of these techniques,thereby allowing one to identify a subject at elevated risk of invasivebreast cancer (through these techniques, for example, and then determineif they should receive radiation therapy or some non-radiation therapytreatment).

In some embodiments, any of the above methods can be combined with anyone or more of the following further aspects as to methods.

In some embodiments, the subject is high risk if they are PR positiveand there is a very high level of FOXA1 (e.g., “elevated risk”). In someembodiments, if a sample is PR positive, then one further analyzes thesample for Ki67, size, or both Ki67 and size. In some embodiments, ifthe sample is PR positive, and FOXA1 negative, then one further analyzesthe sample for a level of Ki67, size, or a level of Ki67 and size,wherein Ki67 positive, a size larger than 5 mm of DCIS, or both, whichindicates high risk (e.g., “elevated risk”). In some embodiments, themethod further comprises analyzing for p16, COX2, and Ki67 in order todetermine elevated risk levels.

In some embodiments, any of the methods and/or compositions fordetermining elevated risk of invasive breast cancer provided in U.S.Pat. Pub. Nos. 20100003189, 20120003639, and 20170350895 can be employedto identify a subject having an elevated risk of invasive breast cancer,the entireties of each of which is hereby incorporated by reference.

In some embodiments, one or more of the denoted markers can be analyzedand/or assayed for. In some embodiments, the markers of HER2 and SIAH2are assayed for separate from other markers. In some embodiments, HER2and SIAH2 are assayed as part of the process for determining elevatedrisk in the subject and/or sample. In some embodiments, HER2 and SIAH2are assayed for with one or more of ER, PR, COX-2, FOXA1, Ki67, and/orp16. In some embodiments, any of the following assays for markers can bedone in combination with HER2 and/or SIAH2, for a subject that has DCIS.

In some embodiments, if the sample is PR positive, one can furtheranalyze the sample for a level of COX-2. In some embodiments, if thesample is PR positive, then one can further analyze the sample for Ki67or size, or both Ki67 and size. In some embodiments, one can analyze thesample for p16 with Ki-67 or p16 with COX-2. In some embodiments, themethod further comprises analyzing at least the following combinations:a) PR, HER2, and SIAH2, b) PR, FOXA1, and COX-2, and c) PR, FOXA1, andKi67. In some embodiments, one also determines if the subject is HER2positive and SIAH2 positive and has DCIS to then determine if thesubject will be receptive to radiation therapy or if a non-radiationtherapy, such as an antibody to HER2, should be employed.

In some embodiments, markers in addition to those disclosed anddescribed herein can be analyzed and/or assayed for. These additionalmarkers are disclosed and described in U.S. patent application Ser. No.12/373,047, filed May 13, 2009 and U.S. patent application Ser. No.13/094,729, filed Apr. 26, 2011, the contents of both applications areincorporated herein by reference in their entirety.

In some embodiments, the method further comprises analyzing at leastCOX-2, Ki67, p16, PR and HER2. In some embodiments, if the sample is PRpositive, one can further analyze the sample for a level of COX-2,wherein COX-2+ with at least FOXA1+ indicates a high risk of invasivebreast cancer. In some embodiments, if the sample is PR positive andthere is a very high level of FOXA1, there is a high risk of invasivebreast cancer. In some embodiments, if the sample is PR positive, thenone can further analyze the sample for Ki67, size, or both Ki67 andsize. In some embodiments, if the sample is PR positive and FOXA1−, onecan further analyze the sample for a level of Ki67, size, or a level ofKi67 and size. Ki67+, a size larger than 5 mm of DCIS, or both,indicates an elevated risk of invasive breast cancer. In someembodiments, the method further comprises analyzing the sample for p16,COX-2, and Ki67. In some embodiments, one also determines if the subjectis HER2 positive and SIAH2 positive and has DCIS to then determine ifthe subject will be receptive to radiation therapy or if a non-radiationtherapy, such as an antibody to HER2, should be employed.

In some embodiments, analysis of each marker is carried out in parallelwith each other. In some embodiments, analysis of each marker is carriedout at overlapping times.

In some embodiments, PR analysis occurs first and any further analysisdepends upon the result of the PR analysis. In some embodiments, thereis no required order for any of the tests and/or analysis for any of themarkers provided herein.

In some embodiments, the method further comprises determining aprognosis of the subject with DCIS. At least a level of SIAH2 and FOXA1relative to the non-DCIS control indicates that the subject has a poorprognosis or wherein no significant difference in the expression ofSIAH2 and FOXA1 relative to a non-tumor control indicates that thesubject has a good prognosis.

In some embodiments, the DCIS sample is further analyzed for COX-2. Insome embodiments, the DCIS sample is further analyzed for p16. In someembodiments, the DCIS sample is analyzed for at least SIAH2, FOXA1, andPR. In some embodiments, the DCIS sample is further analyzed for HER2.In some embodiments, the DCIS sample is further analyzed for COX-2. Insome embodiments, the DCIS sample is further analyzed for Ki67. In someembodiments, the DCIS sample is further analyzed for p16. In someembodiments, one also determines if the subject is HER2 positive andSIAH2 positive and has DCIS to then determine if the subject will bereceptive to radiation therapy or if a non-radiation therapy, such as anantibody to HER2, should be employed.

In some embodiments, the analysis (staining and/or scoring) includesSIAH2 and FOXA1. In some embodiments, the analysis further includes atleast one of the following further combinations: 1) p16, 2) Ki67, 3) PR,4) COX-2, 5) HER2, 6) p16 and Ki67, 7) p16 and PR, 8) p16 and COX-2, 9)p16 and HER2, 10) HER2 and Ki67, 11) HER2 and PR, 12) HER2 and COX-2,13) p16, COX-2, and HER2, 14) HER2, COX-2, and Ki67, 15) HER2, COX-2,and PR, 16) HER2 and COX-2, 17) p16, Ki-67, COX-2, and HER2, 18) Ki-67,HER2, COX-2, and PR, and/or 19) Ki-67, HER2, COX-2, PR, and p16. In someembodiments, one also determines if the subject is HER2 positive andSIAH2 positive and has DCIS to then determine if the subject will bereceptive to radiation therapy or if a non-radiation therapy, such as anantibody to HER2, should be employed.

In some embodiments, the DCIS lesion is further analyzed for grade,necrosis, size, and/or margin status.

In some embodiments, the method further comprises prognosis of a risk byincluding age, menopausal status, mammographic density, tumorpalpability of the subject.

In some embodiments, any of the “PR” steps, methods, compostions, etc.provided herein can be interchanged with an ER step (where ER stainingand/or scoring is performed).

Reports/Recommendations

In some embodiments, any of the present methods can further comprisepreparing a report regarding the risk associated with the human DCIStissue sample. In some embodiments, the report is a written reportproviding the risk of invasive breast cancer. In some embodiments, thereport is generated from and/or includes one or more of the markercombinations provided in Tables 1-9, 11 and 13-15. In some embodiments,the report also details if the subject will be receptive to radiationtherapy or if a non-radiation therapy, such as an antibody to HER2,should be employed.

In some embodiments, any of the present methods further compriseproviding a report regarding a level of risk of a subsequent DCIS event.In some embodiments, the report is a written report providing the riskof a subsequent DCIS event. In some embodiments, the report is generatedfrom and/or includes one or more of the marker combinations provided inTables 1-9, 11 and 13-15.

In some embodiments, the method further comprises recommending atreatment given a result from analyzing the DCIS sample for SIAH2 and atleast one of HER2, PR, FOXA1, or any combination thereof. In someembodiments, the treatment is less aggressive than would have otherwisebeen recommended, without the method predicting a low likelihood ofinvasive breast cancer. In some embodiments, the treatment is moreaggressive than would have otherwise been recommended, without themethod predicting a high likelihood of invasive breast cancer. In someembodiments, the treatment is less aggressive than would have otherwisebeen recommended, without the method predicting a low likelihood of arecurrence of DCIS. In some embodiments, the treatment is moreaggressive than would have otherwise been recommended, without themethod predicting a high likelihood of a recurrence of DCIS. In someembodiments, the report also details if the subject will be receptive toradiation therapy or if a non-radiation therapy, such as an antibody toHER2, should be employed (e.g., depending upon the HER2 and SIAH2results).

In some embodiments, the method further comprises determining a risk ofDCIS, invasive breast cancer, or both. In some embodiments, the methodfurther comprises providing a written report regarding a risk of DCIS,invasive breast cancer, or both (e.g., in line with Tables 1-9, 11 and13-15) as well as whether or not the subject should receive anon-radiation therapy (such as an antibody to HER2) or a radiationtherapy.

Treatment

In some embodiments, any of the above noted methods can include and/orbe followed by an appropriate therapy for the subject, given thesubject's reclassified risk of subsequent DCIS and/or invasive breastcancer and/or responsiveness to radiation therapy (for those in theelevated risk of invasive breast cancer category). In some embodiments,such therapies can be appropriate to reduce a risk of invasive breastcancer, if that is the risk. In some embodiments, for those at anelevated risk of invasive breast cancer, the appropriate treatment ofnon-radiation (for those that are HER2+ and SIAH+) or radiation therapy(for those that are not both HER2+ and SIAH+) can be provided to thesubject or received by the subject. In some embodiments, thenon-radiation therapy is an antibody to HER2, such as trastuzumab.

In some embodiments, a therapy appropriate to reduce a risk of DCISrecurrence comprises at least one of surgical resection, radiationtherapy, anti-hormone therapy. In some embodiments, a therapy can beappropriate if one knows that the subject has a low likelihood of aninvasive event, but would not be appropriate if one knows that thesubject has a high likelihood of an invasive breast cancer event and howlikely the subject is refractory to radiation therapy.

In some embodiments, a therapy appropriate to reduce a risk of invasivebreast cancer comprises at least one of mastectomy, targeted HERstherapy, receptor-targeted chemotherapy. In some embodiments, such atherapy can be appropriate if one knows that the subject has a highlikelihood of an invasive event, but would not be appropriate if oneknows that the subject has a low likelihood of an invasive breast cancerevent. In some embodiments, the therapy is appropriate if the subject isnot, non-responsive to the therapy. In some embodiments, a subject whois predicted to be refractory to radiation therapy will not receive orbe administered a radiation therapy.

In some embodiments, any of the above methods can be followed by“watchful waiting” or other relatively minimal/intrusive therapies. Forexample, when none of the high risk categories are met for invasivebreast cancer, and if the subject has no DCIS risk (or is okay withhaving a DCIS risk), then the approach to treating the DCIS can be totake no immediate action, which can include more frequent breast imagingto provide an early identification of an ipsilateral breast event.

Additional aspects and approaches regarding possible therapeutic actionsthat are specific for the present DCIS subjects are provided below.

Kit

In some embodiments, a kit is provided. The kit can include a FOXA1probe, and a SIAH2 probe. In some embodiments, the kit further comprisesa COX-2 probe, a Ki67 probe, a p16 probe, a PR probe, and a HER2 probe.In some embodiments, the probe is an isolated antibody. In someembodiments, the probe is a nucleic acid that selectively hybridizes toFOXA1, SIAH2, COX-2, Ki67, p16, PR or HER2 as appropriate. In someembodiments, the kit contains enough of the probe and/or the probe issensitive and/or selective enough such that the “+” and “−” states ofone or more of the markers in Tables 1-9, 11 and 13-15 can be adequatelydistinguished from one another. In some embodiments, any of the kitsprovided herein will include at least probes sufficient for HER2 andSIAH. In some embodiments, the HER2 and SIAH2 probes can be part oftheir own kit or performed separately.

In some embodiments, an antibody composition is provided that includesan isolated FOXA1 antibody, and an isolated SIAH2 antibody. In someembodiments, the antibody composition further comprises an isolatedCOX-2 antibody, an isolated Ki67 antibody, an isolated p16 antibody, anisolated PR antibody, and an isolated HER2 antibody. In someembodiments, a HER2 antibody and a SIAH2 antibody are provided incombination with one or more of the other antibodies.

In some embodiments, a solid support comprising probes or antibodiesspecific for at least SIAH2 and FOXA1 is provided. In some embodiments,the probes or antibodies consists essentially of probes or antibodiesspecific for the prediction of DCIS or invasive breast cancer in asubject who has DCIS, including at least HER2 and SIAH. In someembodiments, a solid support comprising probes or antibodies specificfor at least SIAH2 and HER2 is provided.

In some embodiments, the subject and/or sample to be analyzed can be apatient (or from a patient). In some embodiments, the subject has, orhad, DCIS. In some embodiments, the sample came from the DCIS of thesubject in question. There are a variety of ways in which such a subjectcan be identified.

Sample

In some embodiments, the DCIS sample itself can be processed in anynumber of ways to prepare it for screening for the markers. In someembodiments, the DCIS sample has been surgically removed from a patientand preserved. In some embodiments, the DCIS sample is obtained bysurgical removal. In some embodiments, the DCIS sample is cut into oneor more blocks, such as 2, 3, 4, 5 or more blocks.

In some embodiments, a level of SIAH2 and HER2, and/or at least one ofPR, FOXA1, or any combination thereof is at least one of: a RNA level, aDNA level, a protein level. In some embodiments, a level of SIAH2 andHER2 and/or at least one of PR, FOXA1, or any combination thereof is atleast one of: a RNA level, a DNA level, a protein level. In someembodiments, a level of SIAH2 and HER2 and FOXA1 is at least one of: aRNA level, a DNA level, a protein level.

In some embodiments, a signature comprising a level of SIAH2 and HER2,PR, FOXA1, or any combination thereof is at least one of: a RNA level, aDNA level, a protein level. In some embodiments, a signature comprisinga level of SIAH2 and HER2, and at least one of PR or FOXA1 is at leastone of: a RNA level, a DNA level, a protein level. In some embodiments,a signature comprising a level of SIAH2 and HER2 and FOXA1 is at leastone of: a RNA level, a DNA level, a protein level.

In some embodiments, a method of preparing a sample is provided. Themethod comprises obtaining a DCIS sample from a subject and preparing itso that its DNA, RNA, and/or protein can be analyzed for at least SIAH2and HER2 and/or FOXA1.

In some embodiments, the sample is preserved. In some embodiments, thesample is preserved via freezing. In some embodiments, the sample goesthrough (or does not go through) embedding in a chemical such as OptimalCutting Temperature (OCT) compound, or fixation with a chemical(s),including, without limitation, formalin, formaldehyde, quaternaryammonium salts, alcohol, acetone, or other chemicals that preserve orextract DNA, RNA, and/or protein. In some embodiments, the techniqueused is one that allows SIAH2 and HER2 and/or FOXA1 DNA, RNA, and/orprotein to be preserved in an adequate amount and state so that SIAH2and HER2 and/or FOXA1 can be analyzed as provided herein.

In some embodiments, the DCIS sample is processed to allow forimmunohistochemistry of at least SIAH2 and HER2 and/or FOXA1. In someembodiments, at least three such samples (such as in the form of slices)can be prepared).

In some embodiments, analyzing the sample comprises determining anamount of a specified RNA in the sample. The amount of RNA for eachmarker can be determined by any number of techniques, some of which arediscussed elsewhere in the present application. In some embodiments, theRNA level is determined by at least one of: an assay involving nucleicacid microarray, reverse transcriptase-polymerase chain reaction, insitu nucleic acid detection, or a next generation sequencing method. Insome embodiments, expression of at least one of SIAH2 and HER2 and FOXA1is measured by real time quantitative polymerase chain reaction ormicroarray analysis.

In some embodiments, the RNA level is determined by: an assay involvingnucleic acid microarray, reverse transcriptase-polymerase chainreaction, in situ nucleic acid detection, or a next generationsequencing method.

In some embodiments, analyzing the DCIS sample comprises determining anamount of a specified protein in the sample. The amount of protein foreach marker can be determined by any number of techniques, some of whichare discussed elsewhere in the present application. In some embodiments,the protein level is determined by immunohistochemistry,immunofluorescence, or mass spectrometry.

In some embodiments, patient specimens used for the detection of thebiomarkers can be surgically removed breast tissues that are cut intosmall blocks and submerged in fixative. In some embodiments, followingfixation, the blocks can be dehydrated and then embedded in paraffinwax. In some embodiments, the small blocks are no more than 20 mm inlength and 5 mm in thickness to allow complete penetration of thefixative. In some embodiments, the fixation occurs in 10%neutral-buffered formalin for 24 to 48 hours at room temperature topreserve tissue structure and compartmentalization of the variousmarkers. However, other fixatives and fixation times (e.g., 6 to 72hours) can also be compatible with the marker assays. In someembodiments, assays are optimized to use specimens that have been flashfrozen (e.g., in liquid nitrogen), rather than being fixed and embedded.

In some embodiments, the process of sample processing can includedehydration and embedding, which can be done manually or automated witha tissue processing instrument. In either case, the aqueous portion ofthe tissue and the fixation solution can be replaced by passing theblock through a series of increasingly concentrated alcohol solutions.After reaching 100% alcohol, the alcohol is replaced using a chemicallike xylene (or a xylene-free equivalent), followed by introduction ofmolten, low-melting-temperature (e.g., approximately 45° C.) paraffinwax for embedding. The FFPE blocks can be stored for many years prior toanalysis. In some embodiments, “cores” of DCIS tissue can be cut fromthese blocks using a hollow needle and then inserted in an array formatin a separate block of paraffin. Such “tissue microarrays” (TMAs) allowassessment of multiple tissues on a single section/microscope slide.

In some embodiments, ultrathin sections, approximately three to fivemicrometers in thickness, can be cut off the formalin-fixedparaffin-embedded (FFPE) tumor blocks using a microtome. The sectionscan be mounted onto glass microscope slides, ensuring that the tissuedoes not become folded or fragmented, which could interfere with theassays. The glass microscope slides can contain a positively chargedsurface in order bind to the negatively charged tissue sections,although other methods of tissue binding, including adhesives, can alsobe compatible.

In some embodiments, wax removal and rehydration of the tissue sectionscan then be carried out. These processes can be done manually orautomated with certain staining instruments. Wax can be removed from thetissue sections on the slides through heating and/or immersion in asolution of xylene (or an equivalent xylene-free solution, such asNovocastra Bond Dewaxing Solution). Rehydration can be accomplished bypassing the slides through a series of decreasingly concentrated alcoholsolutions until a concentration of 0% is reached (pure water). Followingwax removal and rehydration, the tissue sections can be stained withhematoxylin and eosin (H&E) and for a variety of molecular markers usingimmunohistochemistry (IHC) and/or in situ hybridization (ISH) assays andthen assessed by pathologists or histotechnologists, as described below.The above processing steps can be performed for any of the methodsprovided herein in regard to the various markers (HER2 and SIAH2 and atleast one of COX-2, Ki-67, PR, p16, and FOXA1).

DCIS Diagnosis and Assessment of Pathological Factors—Hematoxylin andEosin Staining

In some embodiments, the subject and/or sample is confirmed as a DCISsample or a subject having DCIS by any of a variety of ways known to oneof skill in the art. This can occur before any of the other method stepsprovided herein (in some embodiments). Provided herein is a set ofnon-exhaustive options for identifying someone with DCIS.

In some embodiments, hematoxylin and eosin (H&E) can be used to stain atleast one tissue section from each patient (or set of arrayed patients)in order to confirm the DCIS diagnosis, assess certain pathologicalfeatures (nuclear grade, architectural pattern[s], and the presence orabsence of necrosis), and as a reference for the interpretation of themolecular marker assays. This histological stain allows thedifferentiation of nuclei and cytoplasm in individual cells, as well asvarious cell types and stromal tissue components, based on the color ofthe staining. The staining can be done manually or automated with aspecial staining instrument. In either case, the section can besubmerged in hematoxylin solution for approximately four minutes tostain the nuclei blue, and then rinsed with tap water (alkaline). Insome embodiments, next, the section can be exposed briefly (typicallyonly a few seconds) to an acid alcohol solution to remove hematoxylinbackground staining, and then rinsed with tap water (alkaline). A“bluing” solution (e.g., lithium carbonate for 30 to 60 seconds) nextmay be applied to enhance the blue color of the hematoxylin in thenuclei, followed by rinsing with water. Eosin solution is then appliedfor approximately two minutes to stain other (eosinophilic) cellularcomponents, followed by rinsing with water. The tissue is dehydratedwith an alcohol series and cleared with xylene (or equivalent), and acover slip is attached using mounting medium. Other options are alsoknown to those of skill in the art. In some embodiments, any method forconfirming the current presence of DCIS can be used on the sample. Insome embodiments, no confirmation process is required. The abovevariables can be altered as appropriate by one of skill in the art.

DCIS itself can be identified in a sample or a subject in a variety ofways. Intraductal proliferative lesions include a group of cytologicallyand architecturally diverse epithelial proliferations originating in theterminal duct lobular unit (TDLU) and can be associated with anincreased risk (of varying magnitude) for the subsequent development ofinvasive breast cancer. DCIS can be regarded as a possible trueprecursor lesion of invasive breast cancer. However, as demonstrated,not all DCIS events go on to form invasive breast cancer. There arevarious grades of DCIS (which, unless otherwise denoted, are allencompassed within the term “DCIS”.

“Low grade DCIS” is composed of small, monomorphic cells, growing in avariety of patterns, including arcades, micropapillae, cribriform orsolid patterns. The nuclei are generally of uniform size and have aregular chromatin pattern with inconspicuous nucleoli with rare mitoticfigures. Low-grade DCIS requires either involvement of two spaces or oneor more duct cross sections exceeding 2 mm in diameter. Althoughdesquamated cells within the ductal lumen may be present, franknecrosis/comedo-type histologic features are not typical for low gradeDCIS. In some embodiments, the definition is the CAP definition.

Cytologic features of DCIS can include: monotonous, uniform rounded cellpopulation; subtle increase in nuclear-cytoplasmic ratio; equidistant orhighly organized nuclear distribution; round nuclei; and hyperchromasiamay or may not be present. Architectural features can include arcades,cribriform, solid and/or micropapillary.

“Intermediate grade DCIS” is often composed of cells cytologicallysimilar to those of low grade DCIS, forming solid, cribriform ormicropapillary patterns, but with some ducts containing intraluminalnecrosis. Others display nuclei of intermediate grade with occasionalnucleoli and coarse chromatin; necrosis may or may not be present.

“High grade DCIS” is usually larger than 5 mm, but even a single <1 mmduct with the typical morphological features is sufficient fordiagnosis. It is composed of highly atypical cells proliferating as onelayer, forming micropapillae, cribriform or solid patterns. Nuclei arehigh grade, markedly pleomorphic, poorly polarized, with irregularcontour and distribution, coarse, clumped chromatin and prominentnucleoli. Mitotic figures are usually common but their presence is notrequired. Comedonecrosis is frequently associated with high grade DCIS,but not necessary for diagnosis. Even a single layer of highlyanaplastic cells lining the duct in a flat fashion is sufficient.

“Atypical Ductal Hyperplasia” is distinct from DCIS. The morphologicalfeatures of atypical ductal hyperplasia are identical to those oflow-grade DCIS, but ADH is limited in size. There are two quantitativecriteria that distinguish ADH from low-grade DCIS: the presence ofhomogeneous involvement of not more than 2 membrane-bound spaces; or asize of ≤2 mm. The use of one or both criteria is considered appropriateby the authors of the WHO classification. In some embodiments, thedefinition is the CAP definition.

Histologic Confirmation of DCIS

In some embodiments, the subject is one who has at least one form ofDCIS. In some embodiments, the presence of DCIS can be confirmed by anyof a variety of techniques, including, for example, using slide-mountedtissue sections stained with hematoxylin and eosin (H&E) or anequivalent histology stain (noted above). In some embodiments, theassessment can be done consistent with WHO classification of tumors ofthe breast (Lakhani S R. WHO classification of tumours of the breast.Lyon: International Agency for Research on Cancer, 2012, and Tavassoli FA, Devilee P. Pathology and genetics of tumours of the breast and femalegenital organs. Lyon: IARC Press, 2003)—see definition of DCIS section.These references contain sample images and review characteristicfeatures of DCIS and differential diagnosis with other breast diseaseentities, such as invasive breast cancer (including microinvasiondefined as invasion ≤1 mm), lobular carcinoma in situ (LCIS), in situPaget's, atypical ductal hyperplasia (ADH), sclerosing adenosis, etc.,the entireties of which are incorporated herein by reference).

In some embodiments, when histological features are not sufficient forthe diagnosis of DCIS, the diagnosis can be confirmed by a secondpathologist. Additional tissue blocks can be employed for morphologicreview.

Cases Suspected to have Invasive (or Microinvasive) Carcinoma Based UponMorphologic Features:

Normal breast ducts and lobules as well as intraductal epithelialproliferations are composed of two epithelial layers. Loss of the outermyoepithelial layer is the hallmark of infiltrating carcinoma of thebreast. The outer myoepithelial layer is retained in all benignproliferative processes as well as ductal carcinoma in situ.Consequently identification of the presence or loss of myoepitheiliumusing antibodies to the myoepithelial-specific proteins can be helpfulin distinguishing in situ from infiltrating carcinoma in circumstanceswhere morphology may be equivocal (Kalof A N et al., Kalof A N, Tam D,Beatty B, Cooper K. Immunostaining patterns of myoepithelial cells inbreast lesions: a comparison of CD10 and smooth muscle myosin heavychain. J Clin Pathol 2004; 57, 625-629; Barbareschi M et al.,Barbareschi M, Pecciarini L, Cangi M G et al. p63, a p53 homologue, is aselective nuclear marker of myoepithelial cells of the human breast. AmJ Surg Pathol; 25, 1054-1060, 2001).

In some embodiments, if there is unequivocal morphologic evidence ofinvasion, including microinvasion, the patient can be considered to beineligible for the prognostic DCIS testing (and will not be tested, orcan be excluded from the assay). In some embodiments, if, uponmorphologic examination of the tumor focus, there is a question ofinvasion of microinvasion, additional myoepithelial marker immunostudies(p63 and/or smooth muscle myosin heavy chain (SMMHC) immunostains) canbe performed to examine the continuity of the myoepithelial cell layersand confirm and/or exclude the presence of (micro)invasive carcinoma. Insome embodiments, other follow procedures can be performed forconfirmation, where appropriate.

Cases Suspected to be of Lobular Origin Based Upon Morphologic Features:

It has been demonstrated that in histologic settings where ductal andlobular neoplasia might be confused, particularly in the setting of insitu carcinoma, where there can be significant differences in patientmanagement, loss of expression of E-cadherin by immunohistochemistry canconfirm the diagnosis of lobular carcinoma, even in the setting ofnon-classical morphologic findings (Acs G et al., Acs G, Lawton T J,Rebbeck T R, LiVolsi V A, Zhang P J. Differential expression ofE-cadherin in lobular and ductal neoplasms of the breast and itsbiologic and diagnostic implications. Am J Clin Pathol 2001; 115, 85-98,2001). In lobular neoplasia, mutations in the E-cadherin gene result inloss of expression of E-cadherin, a cell surface adhesion moleculepresent in normal breast epithelium and ductal carcinoma. The role ofE-cadherin in homotypic cell-cell binding, loss of expression of thiscell surface protein accounts for the characteristic non-cohesive growthpattern of lobular carcinoma.

In some embodiments, if, upon examination of an intraductal epithelialproliferation, it is unclear whether the intraductal tumor is ductal orlobular in nature, an E-cadherin immunostain can be performed to confirmductal or lobular differentiation. In some embodiments, if lobularcarcinoma in situ (LCIS) is confirmed histologically or by loss ofe-cadherin by immunhisotochemistry, the patient could be ineligible forfurther prognostic testing (e.g., will not be tested, or can be excludedfrom the assay). In some embodiments, if the subject currently has LCISand not DCIS, the subject is not treated with the method. In someembodiments, if there is no evidence of DCIS or invasive carcinoma,additional tissue blocks can be requested for morphologic review.

In some embodiments, a sample or subject is excluded from the method ifone or more of the following applies: a) no DCIS identified, b) invasiveor microinvasive carcinoma identified, c) LCIS, not DCIS identified, c)quantitative criteria for low/intermediate DCIS not met: 1) the presenceof homogeneous involvement of more than 2 membrane-bound spaces and/or asize of ≤2 mm. (No quantitative criteria required for high grade DCIS),or 2) tissue folded over in area of interest—not possible to scoreadequately.

Nuclear Grade Determination

In some embodiments, DCIS nuclear grade can be determined by usingslide-mounted tissue sections stained with hematoxylin and eosin (H&E)or an equivalent histology stain. In some embodiments, the assessmentcan be consistent with the College of American Pathologists “Protocolfor the Examination of Specimens from Patients with Ductal Carcinoma inSitu (DCIS) of the Breast” (Lester S C, Bose S, Chen Y Y et al. ArchPathol Lab Med 2009; 133, 15-25), based on references therein. Nucleargrades of I (low), II (intermediate), and/or III (high) will be notedbased on Table 12:

TABLE 12 Grade II Feature Grade I (Low) (Intermediate) Grade III (High)Pleomorphism Monotonous (monomorphic) Intermediate Markedly pleomorphicSize 1.5× to 2× the size of a normal RBC or a Intermediate >2.5× thesize of a normal RBC or a normal duct ep- normal duct epithelial cellnucleus ahelist cell nucleus Chromatin Usually diffuse, finely dispersedchromatin Intermediate Usually vesicular with irregular chromatindistribution Nucleoli Only occasional Intermediate Prominent, oftenmultiple Milsoses Only occasional Intermediate May be frequentOrientation Polarized toward luminal spaces Intermediate Usually notpolarized toward the luminal space * RBC indicates red blood cell.

-   -   Adapted from Lester S et al. Arch Pathol Lab Med 133:15-25 2009.

It is not uncommon to find admixture of various grades of DCIS withinthe same biopsy. In some embodiments, when more than one grade of DCISis present, the proportion (percentage in deciles) of each grade will benoted. In some embodiments subjects with extensive disease and highgrade DCIS will not be considered to be low risk for a subsequentipsilateral breast event. In some embodiments, any of the methodsprovided herein can start with first determining if the subject has DCISand/or the DCIS nuclear grade. In some embodiments, the method does notinclude determining nuclear grade.

Necrosis Determination

In some embodiments, the presence and extent of necrosis in DCIS can beexamined using slide-mounted tissue sections stained with hematoxylinand eosin (H&E) or an equivalent histology stain. The assessment can bedone consistent with the College of American Pathologists “Protocol forthe Examination of Specimens from Patients with Ductal Carcinoma in Situ(DCIS) of the Breast” (June 2012), based on references therein. In someembodiments, necrosis can be classified as follows: A) Not identified:No evidence of necrosis, B) Focal (punctuate): Small foci, indistinct atlow magnification, or single cell necrosis, or C) Central(comedo/extensive): The central portion of an involved ductal space isreplaced by an area of expansive necrosis that is easily detected at lowmagnification. Ghost cells and karyorrhectic debris are generallypresent. Although central necrosis is generally associated withhigh-grade nuclei (comedo DCIS), it can also occur with DCIS of low orintermediate nuclear grade. In some embodiments, any of the methodsprovided herein can include determining necrosis.

IHC Markers Staining/Scoring

When a formalin-based fixation method is used, it creates molecularcross-links in proteins, thereby masking epitopes from recognition byantibodies, and other fixation/preservation methods can also maskepitopes. In such embodiments, epitope retrieval can be a pre-treatmentstep that allows one to unmask the epitopes by reversing, at least inpart, the changes introduced by fixation/preservation. Thus, in someembodiments, any of the methods and/or kits provided herein can includea step or ingredient for epitope retrieval.

Epitope retrieval can be done in different ways by varying the chemicalsin the solution (e.g., buffers, proteolytic enzymes, chelators, etc.),the pH of the solution, the temperature of the solution (e.g., asapplied by a water bath, pressure cooker, autoclave, or microwave oven),and/or the time in the solution, etc. In addition to the specificmethods described in the examples below, many of these other methodscould be used to achieve substantially equivalent results, depending onthe tissue source, primary antibody, and other factors.

Multiple antibodies are commercially available and/or have been reportedin the literature for each protein marker described herein (COX-2, Ki67,HER2, p16, PR, SIAH2, and FOXA1, or those products in Table 0.1), andnew antibodies can also be created. In some embodiments, the antibodiesare raised against and/or recognize different epitopes on the proteinmarkers (COX-2, Ki67, HER2, p16, PR, SIAH2, and FOXA1), and, in othercases, the antibodies are raised against and/or recognize the same (orsimilar) epitopes. The usefulness of an individual antibody in an assaydepends upon its affinity and specificity for the epitope, as well asthe accessibility of the epitope in the assay (e.g., after epitoperetrieval in and IHC assay). Some antibodies recognize more than oneprotein marker and are, therefore, not typically suitable for a specificmarker assay. Other antibodies have low affinity or recognize an epitopethat remains inaccessible in certain samples and are, therefore, notsuitable for certain assay types. For example, an antibody that has ause for an immunoblot of fresh protein lysate may not have utility in anIHC assay on FFPE tissue due to the inability to unmask its epitopethrough epitope retrieval.

The concentrations of primary antibody concentrates commerciallyavailable from manufacturers vary based on the production method (e.g.,tissue culture supernatant, ascites fluid, or whole antiserum), andwhether any purification was done (e.g., affinity purification), butthey are typically in the range of about 0.1 to 10 mg/ml. The optimalfinal primary antibody concentration for incubation on the sectionsdepends on such factors as the binding characteristics of the specificantibody, the incubation time and temperature, and other factors uniqueto the individual laboratory, but it is typically in the range of 0.1 to10 μg/ml, and dilutions ranging from about 1:10 to 1:1,000 are typicallyused. In some embodiments, staining results can be achieved with anantibody over a range of final primary antibody concentrations, as wellas incubation times and temperatures.

In some embodiments, a Novocastra Bond Refine Polymer system can be usedfor detection of the primary antibody. This system includes a polymerbackbone to which multiple secondary antibodies (against rabbit IgG) andenzymes are attached, as well as a rabbit anti-mouse IgG linker (whenused with mouse primary antibodies). The enzymes catalyze a chemicalreaction with DAB to form a brown precipitate that is visualized duringmarker scoring. In some embodiments one of several other detectionmethods can produce adequate results, including systems that utilizeavidin-biotin complex (ABC), labeled streptavidin-biotin (LSAB),catalyzed signal amplification, and/or other technologies that areavailable in a variety of formats from a number of differentmanufacturers. In some embodiments, the ABC and other polymer-basedtechnologies (e.g., Dako EnVision+) can be utilized with similar results(for detection).

In some embodiments, chromogen DAB can be used for final visualizationof the marker through an enzymatic reaction of horseradish peroxidase(HRP) that produces a brown precipitate at the site of the antibodybinding. In some embodiments, HRP can be used in combination with thechromogen 3-amino-9-ethylcarbazole (AEC) to produce red coloration withsubstantially equivalent results. Other options include the enzymealkaline phosphatase in combination with the chromogens nitro bluetetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate(BCIP), and the enzyme glucose oxidase in conjunction with NBT—both ofwhich produce a bluish-purple coloration. There are a variety ofenzyme/chromogen combinations that can produce results.

The following section outlines various representative embodiments forstaining and scoring seven markers (PR, HER2, COX-2, Ki-67, SIAH2,FOXA1, and p16). In some embodiments, other markers that serve the samefunction can be substituted for any one or more of these markers. Insome embodiments ER can be substituted for PR. In some embodiments, anyone or more of these markers can be used in the combinations suggestedin the accompanying tables (Tables 1-9, 11 and 13-15). In someembodiments, one or more of the noted markers can be employed (e.g., forstaining and/or scoring) but a corresponding staining technique and/orcorresponding score is used instead (as outlined herein). In someembodiments, a method for determining whether or not a person is atelevated risk of DCIS, and that involves HER2 and SIAH2 analysis canthen use the same HER2 and SIAH2 data to determine if the subject willbe responsive to radiation therapy or should instead receivenon-radiation therapy, such as a HER2 antibody, such as trastuzumab.

PR Staining

In some embodiments, any technique for PR staining can be used, as longas it is adequate to observe the degree of PR fluctuation provided anddescribed herein. In some embodiments, protein levels can be checked. Insome embodiments, mRNA levels can be checked. In some embodiments, DNAlevels can be checked. In some embodiments, both protein and mRNA levelscan be checked. An elevated level can be a level above that above acontrol or standardized level, for example, a level in a non-DCISsample. Similarly, a lowered level can be a level below a control orstandardized level, for example, a level in a non-DCIS sample. In someembodiments, a “positive” or “elevated” result is one that is above a“negative” or “lowered” result (in the context of scoring).

In some embodiments, to assess progesterone receptor (PR; PGR;HGNC:8910) by IHC, a Leica BOND-MAX automated staining instrument can beused to conduct the following steps with rinsing between each step.Dewaxed and rehydrated tissue sections can be treated with NovocastraPeroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step),followed by Novocastra Bond Epitope Retrieval Solution 1 (based on a 10mM sodium citrate buffer plus 0.05% Tween 20, pH 6.0 solution) for 30minutes at 95° C. to 100° C. (epitope retrieval step). The tissuesections are then incubated at room temperature with mouse monoclonalantibody PgR 636 (Dako M3569) diluted 1:50 in Novocastra PrimaryAntibody Diluent for 30 minutes (primary antibody step), followed byNovocastra Post Primary solution for 15 minutes (rabbit anti-mouse tointroduce IgG linkers), followed by Novocastra Bond Refine Polymer for15 minutes (anti-rabbit poly-HRP-IgG) (secondary detection step),followed by 3,3′-Diaminobenzidine (DAB) for 5 minutes (chromogenvisualization step), followed by <0.1% hematoxylin for 7 minutes(nuclear counterstain step). Finally, a cover slip is attached usingmounting medium. In other embodiments, other options can be employed.

In some embodiments, PR can be detected by a number of primaryantibodies from a number of different manufacturers, and most produceadequate results. In some embodiments, mouse monoclonal 1A6 and rabbitmonoclonal SP2 can be used from Novocastra and Lab Vision. Other optionsinclude mouse monoclonals PgR1294, 16, 1A6, 1E2, Ab-8, Ab-9, hPRa2,hPRa3, and PR88; rabbit monoclonals SP2, SP42, Y85, and EP2; and rabbitpolyclonal A0097 (Dako), as well as many others, from manufacturers likeDako (Agilent), Novocastra (Leica), Ventana Medical Systems (Roche),Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific), BioGenex,Biocare, and Epitomics. In some embodiments, other options can beemployed.

In some embodiments, high pH epitope retrieval (pH 9) can be been donein Tris-EDTA buffer with microwave heating. The titer of each lot ofDako PgR 636 antibody can be adjusted to a reference lot by themanufacturer to ensure consistent staining performance at the samedilution factor, and the 1:50 dilution used in the above example issuggested by the manufacturer. However, a range of dilutions can be used(e.g., 1:10 through 1:500) with similar performance. Alternativedilutions can be optimal with other antibody preparations, and forsituations, some preparations are provided pre-diluted (ready to use).In some embodiments, other options can be employed.

PR Scoring

In some embodiments, any technique for PR scoring can be used, as longas it is adequate to observe the degree of PR fluctuation provided anddescribed herein.

In some embodiments, PR status is determined from the IHC stained slidebased upon the percentage of DCIS tumor cells with nuclear signal. Insome embodiments, all areas of the tissue section containing DCIS can beevaluated to arrive at the percentage. In some embodiments, at leastthree DCIS-containing ducts or 1 mm of DCIS tissue can be employed toscore the markers.

In some embodiments, the intensity of the nuclear signal can be reportedas weak (1+), moderate (2+), or strong (3+). The intensity is theaverage intensity of the DCIS tumor cell nuclei with signal over theentire tissue section relative to the intensity of positive controls runwith the same staining batch. In some embodiments, selection of the DCISregions to be scored and/or the scoring are conducted manually by apathologist and/or automatically using a computer on scanned images ofthe slide.

In some embodiments, a DCIS with less than 10% of tumor cells withnuclear signal can be considered negative, whereas DCIS with greaterthan 10% of tumor cells with nuclear signal can be considered positive,when assayed by the system or examples described herein. In someembodiments, the sample is only considered negative in the presence ofappropriately stained extrinsic and internal controls. In someembodiments, any specimen lacking internal control elements (normalbreast ductal epithelium) that is negative should be reported asuninterpretable (rather than as negative) and repeated using anothertumor specimen from the same or an alternative tumor block.

In some embodiments, alternative thresholds can be applied (e.g., 0%vs. >0%, <1% vs. ≥1%, ≤1% vs. >1%, <5% vs. ≥5%, ≤5% vs. >5%, <10% vs.≥10%, <15% vs. ≥15%, ≤15% vs. >15%, <20% vs. ≥20%, ≤20% vs. >20%, <25%vs. ≥25%, ≤25% vs. >25%, <30% vs. ≥30%, etc. and other thresholds),based upon the technique employed for staining and/or analysis. In someembodiments, alternative techniques and/or scoring methods can be usedfor detection, which can result in a corresponding, but different cutoffrange for high and/or low risk. For such situations, the ranges providedherein for the present technique can be correlated to the othertechnique (for the “corresponding value”) by analyzing the same sample(or two samples from a same DCIS sample) by the two different techniquesand identifying them as being equivalent to one another. Alternativescoring methods, such as the Allred scoring system, immunoscores, andothers that combine the percentage and intensity scoring elements alsoshow effectiveness.

In some embodiments, PR scoring can be as follows: negative is less than5 percent positive by percentage scoring for IHC; positive is greaterthan 10 percent positive by percentage scoring for IHC. In someembodiments, the difference in percent between positive and negative canbe compressed, such that any sample is either positive or negative. Insome embodiments, the difference between positive and negative can bedropped or ignored, if the sample falls within the range. In someembodiments, any of the values between positive and negative can beselected as the absolute distinguishing line between positive andnegative (e.g., 5, 6, 7, 8, 9, or 10).

In some embodiments, FOXA1 scoring can be as follows: negative is lessthan a 100 immunoscore by (intensity times percentage) scoring for IHC;positive is greater than a 100 immonoscore by (intensity timespercentage) scoring for IHC. In some embodiments, the difference inpercent between positive and negative (100) can be compressed, such thatany sample is either positive or negative. In some embodiments, thedifference between positive and negative can be dropped or ignored, ifthe sample falls within the range. In some embodiments, any of thevalues between positive and negative can be selected as the absolutedistinguishing line between positive and negative (e.g., 100 or lower isnegative vs. 100 or higher is positive).

In some embodiments, SIAH2 scoring can be as follows: negative is lessthan 10 percent positive by percentage scoring for IHC; positive isgreater than 20 percent positive by percentage scoring for IHC. In someembodiments, the difference in percent between positive and negative canbe compressed, such that any sample is either positive or negative. Insome embodiments, the difference between positive and negative can bedropped or ignored, if the sample falls within the range. In someembodiments, any of the values between positive and negative can beselected as the absolute distinguishing line between positive andnegative (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, HER2 scoring can be as follows: negative is lessthan a 3+ score by HercepTest scoring criteria for IHC; positive is 3+score by HercepTest scoring criteria for IHC.

In some embodiments, Ki67 scoring can be as follows: negative is lessthan 10 percent positive by percentage scoring for IHC; positive isgreater than 15 percent scoring by percentage for IHC. In someembodiments, the difference in percent between positive and negative canbe compressed, such that any sample is either positive or negative. Insome embodiments, the difference between positive and negative can bedropped or ignored, if the sample falls within the range. In someembodiments, any of the values between positive and negative can beselected as the absolute distinguishing line between positive andnegative (e.g., 10, 11, 12, 13, 14, or 15).

In some embodiments, p16 scoring can be as follows: negative is lessthan 20 percent positive by percentage scoring for IHC; positive isgreater than 25 percent positive by percentage scoring for IHC. In someembodiments, the difference in percent between positive and negative canbe compressed, such that any sample is either positive or negative. Insome embodiments, the difference between positive and negative can bedropped or ignored, if the sample falls within the range. In someembodiments, any of the values between positive and negative can beselected as the absolute distinguishing line between positive andnegative (e.g., 20, 21, 22, 23, 24, or 25).

In some embodiments, COX-2 scoring can be as follows: negative is lessthan 6 by Allred Scoring Criteria for COX-2 for IHC; positive is greaterthan 6 by Allred Scoring Criteria for COX-2 for IHC.

In some embodiments, the above ranges are for IHC assays. In someembodiments, the above ranges are for FISH assays.

In some embodiments, the positive threshold can be higher depending onmarker combinations employed in concert as determined by one skilled inthe art for incorporation into a specific test.

In some embodiments, SIAH2, FOXA1, and COX-2 can further be broken intosub categories of high (very high and high) and low (very low and low).This allows for finer lines to be drawn regarding risk combinations. Insome embodiments, this is simplified for the analysis by having veryhigh and high fall within the “high” grouping and very low and low fallwithin the “low” grouping.

HER2 Staining HER2 IHC

In some embodiments, any technique for HER2 staining can be used, aslong as it is adequate to observe the degree of HER2 fluctuationprovided and described herein. In some embodiments, protein levels canbe checked. In some embodiments, mRNA levels can be checked. In someembodiments, DNA levels can be checked. In some embodiments, bothprotein and mRNA levels can be checked. An elevated level can be a levelabove that above a control or standardized level, for example, a levelin a non-DCIS sample. Similarly, a lowered level can be a level below acontrol or standardized level, for example, a level in a non-DCISsample. In some embodiments, a “positive” or “elevated” result is onethat is above a “negative” or “lowered” result (in the context ofscoring).

In some embodiments, to assess v-erb-b2 avian erythroblastic leukemiaviral oncogene homolog 2 (ERBB2; HGNC:3430; HER2 [human epidermal growthfactor receptor 2]; NEU) by IHC, the following steps can be conductedwith rinsing between each step on a Dako Autostainer (through thechromogen visualization step). Dewaxed and rehydrated tissue sectionscan be treated with Dako Peroxidase Blocking Reagent (peroxidaseblocking step), followed by Dako HercepTest Epitope Retrieval Solution(10 mM citrate buffer plus detergent, pH 6) for 40 minutes in a 95° C.to 99° C. water bath (epitope retrieval step). The tissue sections canthen be incubated at room temperature with pre-diluted rabbit polyclonalanti-HER2 antibody (Dako K5207) for 30 minutes (primary antibody step),followed by Dako HercepTest Visualization Reagent for 30 minutes(secondary detection step), followed by 3,3′-Diaminobenzidine (DAB) for10 minutes (chromogen visualization step), followed by hematoxylin(nuclear counterstain step). Finally, a cover slip is attached usingmounting medium.

In some embodiments, HER2 can be detected by various antibodies to HER2.In some embodiments, mouse monoclonal TAB250 and rabbit monoclonal SP3can be used from Invitrogen and Lab Vision. Other options include mousemonoclonals CB-11, TA9145, Ab-17, 3B5, and PN2A, and rabbit monoclonalsSP3, 4B5, EP1045Y, and EP3, as well as many others, from manufacturerslike Dako (Agilent), Novocastra (Leica), Ventana Medical Systems(Roche), Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific),BioGenex, Biocare, and Epitomics.

In some embodiments, heat-induced epitope retrieval can be performed bymicrowave oven or water bath. In the above example, the HER2 primaryantibody is provided in a pre-diluted (ready to use) format, so nodilution is performed. However, other preparations of HER2 primaryantibody are available in concentrated format requiring dilution. Forexample, a 200 μg/ml Ig concentrate may be diluted 1:400 to 1:800 to afinal Ig concentration of 0.25 to 0.50 μg/ml, or an alternativeconcentrate may have an optimal dilution of 1:100 with similar resultsachieved in a dilution range of 1:50 to 1:500.

HER2—ISH

In some embodiments, an alternative method to assess HER2 can be ISH. Afluorescent ISH (FISH) assay using a Vysis PathVysion HER-2 DNA ProbeKit (Abbott Molecular) can include the following steps with rinsingbetween each step. The dewaxed tissue section can be treated with 0.2 NHCl for 20 minutes and then Vysis Pretreatment Solution (1 N sodiumisothiocyanate) for 30-60 minutes at 80° C. (pretreatment step),followed by Vysis Protease Solution for 10-60 minutes at 37° C.(protease step), followed by 10% neutral buffered formalin (fixationstep), followed by a series of increasing concentration alcoholsolutions (dehydration step).

In some embodiments, a DNA probe mixture is applied to the tissuesection under a sealed coverslip. The mixture contains a Locus SpecificIdentifier for HER-2 (a 190-Kb Spectrum Orange directly-labeled,fluorescent DNA probe specific for the HER-2 gene locus at 17q11.2-q12)and a Chromosome Enumeration Probe for chromosome 17 (CEP17; a 5.4 KbSpectrum Green directly-labeled, fluorescent DNA probe specific for thealpha satellite DNA sequence at the centromeric region of chromosome 17at 17p11.1-q11.1), as well as unlabeled blocking DNA to suppresssequences contained within the target loci that are common to otherchromosomes. The slide is then placed into a Thermobrite instrument andsubjected to a temperature of 73° C. for 5 minutes (DNA denaturationstep) followed by an incubation for 14-24 hours at 37° C. (DNAhybridization step).

In some embodiments, the tissue is treated with two washes of 2× salinesodium citrate (SSC) plus 0.3% NP-40, the first for 2-5 minutes at roomtemperature, and the second for 2 minutes at 71-73° C. (posthybridization wash step). Finally, the nuclei are stained blue with a4,6 diamidino-2-phenylindole (DAPI) solution (counterstaining step).

In some embodiments, an alternative ISH method based on silverdeposition (SISH; Ventana Medical Systems Inform HER2 kit) can be usedaccording to manufacturer instructions with substantially equivalentresults. In some embodiments, other HER2 ISH methods, for examplechromogenic ISH (CISH), can also be employed.

HER2 Scoring—HER2 IHC Scoring

In some embodiments, any technique for HER2 scoring can be used, as longas it is adequate to observe the degree of HER2 fluctuation provided anddescribed herein.

In some embodiments, HER2 status is determined from the IHC stainedslide according to College of American Pathologists-American Society ofClinical Oncology (CAP-ASCO) guidelines (Wolff et al. J Clin Oncol31:3997, 2013) with modification for scoring on intraductal tumor cells.HER2 scores of 0-3+ are defined as follows: 0 is defined by no stainingobserved or membrane staining that is incomplete and is faint/barelyperceptible and within ≤10% of the tumor cells. 1+ is defined byincomplete membrane staining that is faint/barely perceptible andwithin >10% of the tumor cells. 2+ is defined by circumferentialmembrane staining that is incomplete and/or weak/moderate (observed in ahomogeneous and contiguous population) and within >10% of the tumorcells, or complete and circumferential membrane staining that is intenseand within ≤10% of the tumor cells. 3+ is defined by circumferentialmembrane staining that is complete and intense (observed in ahomogeneous and contiguous population and within >10% of the tumorcells, readily appreciated using a low power objective).

In some embodiments, selection of the DCIS regions to be scored and/orthe scoring are conducted manually by a pathologist and/or automaticallyusing a computer on scanned images of the slide. In some embodiments,results of 0, 1+, or 2+ are considered negative, and 3+ is consideredpositive. In some embodiments, 2+ results could be considered equivocal,triggering a HER2 ISH assay to determine status through quantitation ofHER2 gene amplification (amplified patients are positive). In someembodiments, HER2 IHC could be completely replaced by a HER2 ISH assay.Both of these alternative approaches have shown utility in relatedstudies.

HER2 ISH Scoring

In some embodiments, HER2 status is determined from a FISH stained slideby counting the orange HER2 and green CEP17 signals in a minimum of 20DCIS cell nuclei and then calculating the ratio. The DCIS is consideredHER2 non-amplified (negative) when there is an equal number of orangeand green signals or the ratio of Orange to green is less than 2.0 withan average HER2 copy number per cell of less than 4.0. The DCIS isconsidered HER2 amplified (positive) when the ratio of orange to greensignals is greater than 2.0. Cells are also considered amplified(positive) when the ratio of orange to green signals is less than 2.0with an average HER2 copy number per cell greater than or equal to 6.0.When the ratio of orange to green signals is less than 2.0 with anaverage HER2 copy number per cell of greater than or equal to 4.0 andless than 6.0, 20 additional cells are counted, and the ratio isre-calculated for all 40 cells, and the threshold of <2.0 (negative) vs.≥2.0 (positive) is applied.

Ki-67 Staining

In some embodiments, any technique for Ki-67 staining can be used, aslong as it is adequate to observe the degree of Ki-67 fluctuationprovided and described herein. In some embodiments, protein levels canbe checked. In some embodiments, mRNA levels can be checked. In someembodiments, DNA levels can be checked. In some embodiments, bothprotein and mRNA levels can be checked. An elevated level can be a levelabove that above a control or standardized level, for example, a levelin a non-DCIS sample. Similarly, a lowered level can be a level below acontrol or standardized level, for example, a level in a non-DCISsample. In some embodiments, a “positive” or “elevated” result is onethat is above a “negative” or “lowered” result (in the context ofscoring).

In some embodiments, Ki-67 (MKI67; MIB-1; HGNC:7107) levels can beassessed by IHC, a Leica BOND-MAX automated staining instrument is usedto conduct the following steps with rinsing between each step. In someembodiments, dewaxed and rehydrated tissue sections are treated withNovocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blockingstep), followed by Novocastra Bond Epitope Retrieval Solution 2 (basedon a 10 mM Tris Base and 1 mM ethylenediaminetetraacetic acid [EDTA]buffer plus 0.05% Tween 20, pH 9.0 solution) for 30 minutes at 95° C. to100° C. (epitope retrieval step). In some embodiments, the tissuesections are then incubated at room temperature with mouse monoclonalantibody MIB-1 (Dako M7240) diluted 1:50 in Novocastra Primary AntibodyDiluent for 30 minutes (primary antibody step), followed by NovocastraPost Primary solution for 15 minutes (rabbit anti-mouse IgG to introduceIgG linkers), followed by Novocastra Bond Refine Polymer for 15 minutes(anti-rabbit poly-HRP-IgG) (secondary detection step), followed by3,3′-Diaminobenzidine (DAB) for 5 minutes (chromogen visualizationstep), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstainstep). Finally, a cover slip is attached using mounting medium.

In some embodiments, Ki-67 can be detected by any Ki-67 specificantibody. In some embodiments, the antibody can be mouse monoclonalsMM1, K-2; 7B11, BGX-297, Ki88, Ki-S5, and DVB-2; rabbit monoclonals SP6,30-9, EPR3611; and rabbit polyclonal NCL-Ki67p, as well as many others,from manufacturers like Dako (Agilent), Novocastra (Leica), VentanaMedical Systems (Roche), Cell Marque (Sigma-Aldrich), Lab Vision (ThermoScientific), BioGenex, and Biocare.

In some embodiments, a dilution of 1:50 is used for Dako MIB-1 antibody,although other dilutions, e.g., 1:75 to 1:150, can be used, includingdilutions in the range of 1:40 to 1:600. Similarly, other antibodymanufacturers report recommended dilutions in the range of 1:100 to1:200 for their antibody preparations, and some preparations areprovided pre-diluted (ready to use).

Ki-67 Scoring

In some embodiments, any technique for Ki-67 scoring can be used, aslong as it is adequate to observe the degree of Ki-67 fluctuationprovided and described herein.

In some embodiments, Ki-67 status is determined from the IHC stainedslide based upon the percentage of DCIS tumor cells with nuclear signal.In some embodiments, all areas of the tissue section containing DCIS areevaluated to arrive at the percentage. In some embodiments, at leastthree DCIS-containing ducts or 1 mm of DCIS tissue are used to score themarkers. In some embodiments, the intensity of the signal is alsoreported as weak (1+), moderate (2+), or strong (3+). In someembodiments, the intensity is the average intensity of the DCIS tumorcell nuclei with signal over the entire tissue section. In someembodiments, selection of the DCIS regions to be scored and/or thescoring are conducted manually by a pathologist and/or automaticallyusing a computer on scanned images of the slide.

In some embodiments, DCIS with less than 10% of tumor cells with nuclearsignal is considered negative, whereas DCIS with greater than 10% oftumor cells with nuclear signal is considered positive. The sample isonly considered negative in the presence of an appropriately stainedpositive control.

In some embodiments, alternative thresholds (e.g., 0% vs. >0%, <1% vs.≥1%, ≤1% vs. >1%, <5% vs. ≥5%, ≤5% vs. >5%, <10% vs. ≥10%, <15% vs.≥15%, ≤15% vs. >15%, <20% vs. ≥20%, ≤20% vs. >20%, <25% vs. ≥25%, ≤25%vs. >25%, <30% vs. ≥30%, etc. can be employed based upon the techniqueemployed for staining and/or analysis. In some embodiments, alternativetechniques and/or scoring methods can be used for detection, which canresult in a corresponding, but different cutoff range for high and/orlow risk. For such situations, the ranges provided herein for thepresent technique can be correlated to the other technique (for the“corresponding value”) by analyzing the same sample (or two samples froma same DCIS sample) by the two different techniques and identifying themas being equivalent to one another. Alternative scoring methods, such asthe Allred scoring system, immunoscores, and others that combine thepercentage and intensity scoring elements also show effectiveness.

p16/INK4A Staining

In some embodiments, any technique for p16 staining can be used, as longas it is adequate to observe the degree of p16 fluctuation provided anddescribed herein. In some embodiments, protein levels can be checked. Insome embodiments, mRNA levels can be checked. In some embodiments, DNAlevels can be checked. In some embodiments, both protein and mRNA levelscan be checked. An elevated level can be a level above that above acontrol or standardized level, for example, a level in a non-DCISsample. Similarly, a lowered level can be a level below a control orstandardized level, for example, a level in a non-DCIS sample. In someembodiments, a “positive” or “elevated” result is one that is above a“negative” or “lowered” result (in the context of scoring).

In some embodiments, to assess the p16 isoform of cyclin-dependentkinase inhibitor 2A (p16/INK4A; CDKN2A; MTS1; HGNC:1787) by IHC, a LeicaBOND-MAX automated staining instrument can be used to conduct thefollowing steps with rinsing between each step. In some embodiments, itcan be dewaxed and rehydrated tissue sections are treated withNovocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blockingstep), followed by Novocastra Bond Epitope Retrieval Solution 2 (basedon a 10 mM Tris Base and 1 mM EDTA buffer plus 0.05% Tween 20, pH 9.0solution) for 30 minutes at 95° C. to 100° C. (epitope retrieval step).The tissue sections can then be incubated at room temperature withpre-diluted mouse monoclonal antibody E6H4 (Ventana Medical SystemsCINtec p16 Histology Kit) for 30 minutes (primary antibody step),followed by Novocastra Post Primary solution for 15 minutes (rabbitanti-mouse IgG to introduce IgG linkers), followed by Novocastra BondRefine Polymer for 15 minutes (anti-rabbit poly-HRP-IgG) (secondarydetection step), followed by 3,3′-Diaminobenzidine (DAB) for 5 minutes(chromogen visualization step), followed by <0.1% hematoxylin for 7minutes (nuclear counterstain step). In some embodiments, a cover slipis attached using mounting medium.

In some embodiments, p16/INK4A can be detected by a primary antibody. Insome embodiments, mouse monoclonal DCS-50.1/A7 (Neomarkers) can be used.In some embodiments, mouse monoclonals 6H12, JC8, 16PO4; 16PO7, andG175-405 and rabbit monoclonal EPR1473, as well as others can be used.In some embodiments, the p16/INK4A primary antibody is provided in apre-diluted (ready to use) format, so no dilution is performed. However,other preparations of p16/INK4A primary antibody are available inconcentrated format, and dilutions are recommended by the manufacturersin a range of 1:75 to 1:500 or even 1:25 to 1:800, depending on thespecific protocol.

p16/INK4A Scoring

In some embodiments, any technique for p16 scoring can be used, as longas it is adequate to observe the degree of p16 fluctuation provided anddescribed herein.

In some embodiments, p16/INK4A status is determined from the IHC stainedslide based upon the percentage of DCIS tumor cells with nuclear signal,qualified by the intensity of the signal. In some embodiments, theintensity of the signal is reported as weak (1+), moderate (2+), orstrong (3+). In some embodiments, the intensity is the average intensityof the DCIS tumor cell nuclei with signal over the entire tissue sectionrelative to the intensity of positive controls run with the samestaining batch. In some embodiments, cells with nuclear signal of atleast intermediate (2+) intensity are considered positive. In someembodiments, cells with absent or weak (1+) staining are considerednegative. In some embodiments, all areas of the tissue sectioncontaining DCIS are evaluated to arrive at the percentage. In someembodiments, at least three DCIS-containing ducts or 1 mm of DCIS tissuecan be used to score the markers. In some embodiments, selection of theDCIS regions to be scored and/or the scoring are conducted manually by apathologist and/or automatically using a computer on scanned images ofthe slide.

In some embodiments, DCIS with less than or equal to 25% of tumor cellswith nuclear signal of at least moderate intensity is considerednegative, whereas DCIS with greater than 25% of tumor cells with nuclearsignal of at least moderate intensity is considered positive. The sampleis only considered negative in the presence of an appropriately stainedpositive control. Alternative thresholds (e.g., <20% vs. ≥20%, ≤20%vs. >20%, <25% vs. ≥25%, <30% vs. ≥30%, ≤30% vs. >30%, <40% vs. ≥40%,etc. and other thresholds) can be employed based upon the techniqueemployed for staining and/or analysis. In some embodiments, alternativetechniques and/or scoring methods can be used for detection, which canresult in a corresponding, but different cutoff range for high and/orlow risk. For such situations, the ranges provided herein for thepresent techniques can be correlated to the other technique (for the“corresponding value”) by analyzing the same sample (or two samples froma same DCIS sample) by the two different techniques and identifying themas being equivalent to one another. Alternative scoring methods, such asthe Allred scoring system, immunoscores, and others that combine thepercentage and intensity scoring elements also show effectiveness.

The staining pattern of p16/INK4A can be nuclear and cytoplasmic and isoften heterogeneous in nature. In addition, p16/INK4A staining can bepresent in both the DCIS tumor cells and the surrounding stromal cells.

COX-2 Staining

In some embodiments, any technique for COX-2 staining can be used, aslong as it is adequate to observe the degree of COX-2 fluctuationprovided and described herein. In some embodiments, protein levels canbe checked. In some embodiments, mRNA levels can be checked. In someembodiments, DNA levels can be checked. In some embodiments, bothprotein and mRNA levels can be checked. An elevated level can be a levelabove that above a control or standardized level, for example, a levelin a non-DCIS sample. Similarly, a lowered level can be a level below acontrol or standardized level, for example, a level in a non-DCISsample. In some embodiments, a “positive” or “elevated” result is onethat is above a “negative” or “lowered” result (in the context ofscoring).

In some embodiments, prostaglandin-endoperoxide synthase 2 (PTGS2;cyclooxygenase-2 [COX-2]; HGNC:9605) can be assessed by IHC; a LeicaBOND-MAX automated staining instrument is used to conduct the followingsteps with rinsing between each step. In some embodiments, dewaxed andrehydrated tissue sections are treated with Novocastra Peroxide Block(3-4% hydrogen peroxide) (peroxidase blocking step), followed byNovocastra Bond Epitope Retrieval Solution 1 (based on a 10 mM sodiumcitrate buffer plus 0.05% Tween 20, pH 6.0 solution) for 30 minutes at95° C. to 100° C. (epitope retrieval step). In some embodiments, thetissue sections are then incubated at room temperature with rabbitmonoclonal antibody SP21 (Cell Marque 240R-16) diluted 1:50 inNovocastra Primary Antibody Diluent for 30 minutes (primary antibodystep), followed by Novocastra Bond Refine Polymer for 15 minutes(anti-rabbit poly-HRP-IgG) (secondary detection step), followed by3,3′-Diaminobenzidine (DAB) for 5 minutes (chromogen visualizationstep), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstainstep). Finally, a cover slip is attached using mounting medium.

In some embodiments, COX-2 can be detected by primary antibodies. Insome embodiments, mouse monoclonal CX-294 (Dako) can be used. In someembodiments, mouse monoclonal 4H12 (Novocastra) and rabbit monoclonalSP21 from manufacturers like Ventana Medical Systems (Roche), CellMarque (Sigma-Aldrich), Lab Vision (Thermo Scientific), and Biocare canbe used.

In some embodiments, a dilution of 1:50 can be used for Cell Marque SP21antibody. In some embodiments, a dilution of 1:100 to 1:500, or in therange of 1:50 to 1:500, 1:50 to 1:200 can be used. In addition, somepreparations are provided in pre-diluted (ready to use) form.

COX-2 Scoring

In some embodiments, any technique for COX-2 scoring can be used, aslong as it is adequate to observe the degree of COX-2 fluctuationprovided and described herein.

In some embodiments, COX-2 status can be determined from the IHC stainedslide based upon the percentage of DCIS tumor cells with cytoplasmicsignal and the intensity of the signal, in the form of an Allred score.In some embodiments, the intensity is reported as absent (0), weak (1),intermediate (2), or strong (3) and represents the average signalintensity over the entire tissue section relative to the intensity ofpositive controls run with the same staining batch. In some embodiments,the percentage is converted to a proportion score as follows: 0, 0%positive; 1, ≤1% positive; 2, >1-10% positive; 3, 11-33% positive; 4,34-66% positive; 5, 67-100% positive. The Allred score is the sum of theintensity and proportion scores on a scale of 0-8.

In some embodiments, all areas of the tissue section containing DCIS areevaluated. In some embodiments, at least three DCIS-containing ducts or1 mm of DCIS tissue is employed to score the markers. Selection of theDCIS regions to be scored and/or the scoring can be conducted manuallyby a pathologist and/or automatically using a computer on scanned imagesof the slide.

In some embodiments, a DCIS with an Allred score 0 to 6 is considerednegative, whereas DCIS with an Allred score of 7 or 8 is consideredpositive. In some embodiments, the sample is considered negative in thepresence of an appropriately stained positive control. In someembodiments, alternative techniques and/or scoring methods can be usedfor detection, which can result in a corresponding, but different cutoffrange for high and/or low risk. For such situations, the ranges providedherein for the present techniques can be correlated to the othertechnique (for the “corresponding value”) by analyzing the same sample(or two samples from a same DCIS sample) by the two different techniquesand identifying them as being equivalent to one another. In someembodiments, an alternative scoring method, such as the Allred scoringsystem, immunoscores, and others that combine the percentage andintensity scoring elements also show effectiveness.

FOXA1 Staining

In some embodiments, any technique for FOXA1 staining can be used, aslong as it is adequate to observe the degree of FOXA1 fluctuationprovided and described herein. In some embodiments, protein levels canbe checked. In some embodiments, mRNA levels can be checked. In someembodiments, DNA levels can be checked. In some embodiments, bothprotein and mRNA levels can be checked. An elevated level can be a levelabove that above a control or standardized level, for example, a levelin a non-DCIS sample. Similarly, a lowered level can be a level below acontrol or standardized level, for example, a level in a non-DCISsample. In some embodiments, a “positive” or “elevated” result is onethat is above a “negative” or “lowered” result (in the context ofscoring).

In some embodiments, to assess forkhead box A1 (FOXA1; HGNC:5021) byIHC, a Leica BOND-MAX automated staining instrument can be used toconduct the following processes with rinsing between each step. In someembodiments, dewaxed and rehydrated tissue sections can be treated withNovocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blockingstep), followed by Novocastra Bond Epitope Retrieval Solution 2 (basedon a 10 mM Tris Base and 1 mM ethylenediaminetetraacetic acid [EDTA]buffer plus 0.05% Tween 20, pH 9.0 solution) for 30 minutes at 95° C. to100° C. (epitope retrieval step). The tissue sections can then beincubated at room temperature with mouse monoclonal antibody 2F83 (CellMarque 405M-16) diluted 1:25 in Novocastra Primary Antibody Diluent for30 minutes (primary antibody step), followed by Novocastra Post Primarysolution for 15 minutes (rabbit anti-mouse IgG to introduce IgGlinkers), followed by Novocastra Refine Polymer for 15 minutes(anti-rabbit poly-HRP-IgG) (secondary detection step), followed by3,3′-Diaminobenzidine (DAB) for 5 minutes (chromogen visualizationstep), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstainstep). A cover slip can be attached using mounting medium.

In some embodiments, FOXA1 mouse monoclonal antibody 2F83 from Abcam canbe used at a dilution of 1:450 (or, for example, 1:2,000) with effectiveresults or used at a dilution of 1:25 (manufacturer recommended startingdilution range of 1:25 to 1:100, based on an initial estimated Igconcentration of 2.5 to 25.0 μg/ml with a final estimated optimal Igconcentration range of 0.1 to 1.0 μg/ml). In some embodiments, mousemonoclonal antibodies can include 2F83, 3A8, 1B1, 2D7, 3C1, and 4F6, andrabbit monoclonals SP88 and EPR10881 from Thermo Scientific, SpringBioscience, Epitomics, Millipore, and a variety of other manufacturers.Various dilutions can be used with other antibody preparations (e.g.,Millipore recommends a 1:500 dilution of their 1 mg/ml version of 2F83,and Thermo Scientific recommends a 1:20 to 1:200 dilution of their 1mg/ml preparation of clone 3A8). And some FOXA1 antibody preparationsare provided pre-diluted (ready to use).

FOXA1 Scoring

In some embodiments, any technique for FOXA1 scoring can be used, aslong as it is adequate to observe the degree of FOXA1 fluctuationprovided and described herein.

In some embodiments, FOXA1 status can be determined from the IHC stainedslide based upon the percentage of DCIS tumor cells with nuclear signaland the intensity of the signal, in the form of an immunoscore. Theintensity can be reported as absent (0), weak (1), intermediate (2), orstrong (3) and can represent the average signal intensity over theentire tissue section relative to the intensity of positive controls runwith the same staining batch. In some embodiments, the immunoscore canbe the product of the intensity and percentage scores on a scale of0-300.

In some embodiments, all areas of the tissue section containing DCIS areevaluated. In some embodiments, three DCIS-containing ducts or 1 mm ofDCIS tissue is used to score the markers. Selection of the DCIS regionsto be scored and/or the scoring can be conducted manually by apathologist and/or automatically using a computer on scanned images ofthe slide.

In some embodiments, DCIS with an immunoscore less than 100 isconsidered FOXA1 low, DCIS with an immunoscore between 100 and 250 isconsidered FOXA1 intermediate, and DCIS with an immunoscore greater than250 is considered FOXA1 high. Alternative lower (e.g., 40 or 150) and/orupper (e.g., 150 or 250) thresholds, as well as alternative scoringmethods, also show utility. In addition, the utility can vary based onthe outcome being predicted (i.e., a DCIS or invasive event in theipsilateral breast). In some embodiments, FOXA1 is merely treated aseither being “negative” (100 or lower) or positive (greater than 100).

In some embodiments, alternative techniques and/or scoring methods canbe used for detection, which can result in a corresponding, butdifferent cutoff range for high and/or low risk. For such situations,the ranges provided herein for the present techniques can be correlatedto the other technique (for the “corresponding value”) by analyzing thesame sample (or two samples from a same DCIS sample) by the twodifferent techniques and identifying them as being equivalent to oneanother. In some embodiments, an alternative scoring method, such as theAllred scoring system, immunoscores, and others that combine thepercentage and intensity scoring elements also show effectiveness.

SIAH2 Staining

In some embodiments, any technique for SIAH2 staining can be used, aslong as it is adequate to observe the degree of SIAH2 fluctuationprovided and described herein. In some embodiments, protein levels canbe checked. In some embodiments, mRNA levels can be checked. In someembodiments, DNA levels can be checked. In some embodiments, bothprotein and mRNA levels can be checked. An elevated level can be a levelabove a control or standardized level, for example, a level in anon-DCIS sample. Similarly, a lowered level can be a level below acontrol or standardized level, for example, a level in a non-DCISsample. In some embodiments, a “positive” or “elevated” result is onethat is above a “negative” or “lowered” result (in the context ofscoring).

In some embodiments, to assess SIAH2 E3 ubiquitin protein ligase 2(SIAH2; seven in absentia [Drosophila] homolog 2; HGNC:10858) by IHC, aLeica BOND-MAX automated staining instrument can be used to conduct thefollowing steps with rinsing between each step. In some embodiments,dewaxed and rehydrated tissue sections are treated with NovocastraPeroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step),followed by Novocastra Bond Epitope Retrieval Solution 1 (based on a 10mM sodium citrate buffer plus 0.05% Tween 20, pH 6.0 solution) for 30minutes at 95° C. to 100° C. (epitope retrieval step). In someembodiments, the tissue sections are then incubated at room temperaturewith mouse monoclonal antibody 24E6H3 (Santa Cruz Biotechnologysc-81787) diluted 1:200 in Novocastra Primary Antibody Diluent for 30minutes (primary antibody step), followed by Novocastra Post Primarysolution for 15 minutes (rabbit anti-mouse to introduce IgG linkers),followed by Novocastra Bond Refine Polymer for 15 minutes (anti-rabbitpoly-HRP-IgG) (secondary detection step), followed by3,3′-Diaminobenzidine (DAB) for 5 minutes (chromogen visualizationstep), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstainstep). In some embodiments, a cover slip is attached using mountingmedium.

In some embodiments, epitope retrieval can be done at a higher pH (8.0)in EDTA-containing buffer in either a 98° C. water bath or steam chamberwith both mouse monoclonal antibody 24E6H3 and its parental clone 24E6in related studies. In some embodiments, alternative antibodies can beused, including those from Novus Biologicals. Different dilutions can beused with different preparations of these antibody, including dilutionswithin the range of 1:40 to 1:200. In some embodiments, other primaryantibodies can be used, such as mouse monoclonal 35F7I4 (CreativeDiagnostics), which works for IHC (recommended dilution of 1:40 to1:50), and mouse monoclonals 1F5, 2G6, and SIAH2-369 (available fromAbnova, Epigentek, US Biologicals, Sigma-Aldrich, and CreativeDiagnostics).

SIAH2 Scoring

In some embodiments, any technique for SIAH2 scoring can be used, aslong as it is adequate to observe the degree of SIAH2 fluctuationprovided and described herein.

In some embodiments, SIAH2 status is determined from the IHC stainedslide based upon the percentage of DCIS tumor cells with nuclear signal.In some embodiments, all areas of the tissue section containing DCIS areevaluated to arrive at the percentage. In some embodiments, at leastthree DCIS-containing ducts or 1 mm of DCIS tissue is used to score themarkers. In some embodiments, the intensity of the signal can bereported as weak (1+), moderate (2+), or strong (3+). In someembodiments, the intensity is the average intensity of the DCIS tumorcell nuclei with signal over the entire tissue section. In someembodiments, selection of the DCIS regions to be scored and/or thescoring are conducted manually by a pathologist and/or automaticallyusing a computer on scanned images of the slide.

In some embodiments, DCIS with less than 20% of tumor cells with nuclearsignal is considered negative, whereas DCIS with greater than or equalto 20% of tumor cells with nuclear signal is considered positive.

In some embodiments, the sample is only considered negative in thepresence of an appropriately stained positive control.

In some embodiments, alternative techniques and/or scoring methods canbe used for detection, which can result in a corresponding, butdifferent cutoff range for high and/or low risk. For such situations,the ranges provided herein for the noted techniques can be correlated tothe other technique (for the “corresponding value”) by analyzing thesame sample (or two samples from a same DCIS sample) by the twodifferent techniques and identifying them as being equivalent to oneanother. In some embodiments, an alternative scoring method, such as theAllred scoring system, immunoscores, and others that combine thepercentage and intensity scoring elements also show effectiveness. Insome embodiments, alternative thresholds (e.g., ≤20% vs. >20%, <25% vs.≥25%, ≤25% vs. >25%, <30% vs. ≥30%, ≤30% vs. >30%, <40% vs. ≥40%, ≤40%vs. >40%, etc. and other thresholds) can be used. In some embodiments,alternative scoring methods, such as the Allred scoring system,immunoscores, and others that combine the percentage and intensityscoring elements can also be used.

An example of SIAH2 staining and scoring is presented in FIG. 3. FIG. 3depicts SIAH2 IHC assays (top, negative, on a UUH TMA; bottom, positive,on a Biomax BR8011 TMA).

Statistical Analysis

Kaplan-Meier survival analyses can be used to estimate the proportionsof patients who experienced first events (DCIS or invasive recurrence)after initial DCIS diagnosis/surgery. Hazard ratios (HR) can bedetermined using Cox proportional hazards analysis. At the time of firstevents (DCIS or invasive), patients can be censored for the other eventtype. Patients can also be censored if an event is detected within 6months of surgery or on the first post-surgery mammogram, because thiscan be considered as persistent, rather than recurrent, disease. In someembodiments, subjects with persistent disease are excluded from thevarious methods provided herein.

Methods of Assessing Markers

While the above noted assaying system and scoring systems have been putforth, in some embodiments, alternative techniques and/or scoringmethods can be used for detection, which can result in a corresponding,but different cutoff range for high and/or low risk. For suchsituations, the ranges provided herein for the present techniques can becorrelated to the other technique (for the “corresponding value” of oneor more of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and/or HER2) by analyzingthe same sample (or two samples from a same DCIS sample) by the twodifferent techniques and identifying them as being matched or equal toone another. In some embodiments, an alternative scoring method, such asthe Allred scoring system, immunoscores, and others that combine thepercentage and intensity scoring elements also show effectiveness. Insome embodiments, there can be a corresponding technique and/or scorefor one or more of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and/or HER2. Insome embodiments, there can be a corresponding technique for one or moreof COX-2, Ki67, p16, SIAH2, FOXA1, PR, and/or HER2. In some embodiments,there can be a corresponding score for one or more of COX-2, Ki67, p16,SIAH2, FOXA1, PR, and/or HER2. As will be appreciated by one of skill inthe art, these corresponding scores and/or techniques can be used forany of the embodiments provided herein, and are expressly contemplatedas alternatives for each and all disclosure regarding the noted markers.

In some embodiments, protein detection assays can be used, including forexample, immunohistochemistry, immunofluorescence, mass spectrometry, orothers, discussed in more detail below. In some embodiments, mRNAdetection assays can be used, including, for example, nucleic acidhybridization-based methods such as Northern blots, gene expressionarrays, quantitative real-time polymerase chain reaction (qPCR),nCounter, in situ nucleic acid detection, etc., as well asnext-generation RNA sequencing (RNA-Seq)), and others. In someembodiments, techniques to detect changes at the DNA level can be used,including, for example, polymerase chain reaction (PCR), in situhybridization (ISH), next generation sequencing (NGS), and others aswill be readily understood by those of skill in the art.

In some embodiments, while corresponding values (involving alternativescoring or alternative assays to analyze the sample) are used, therelative result (for example, high vs. low or very high vs. medium vs.low) will be maintained between the various techniques for analysis orscoring systems. Thus, scoring “high” in one system will be correlatedto scoring “high” in another system, without significant complicationsor difficulties. Thus, various results can be ported from one system toanother, as desired, as long as the levels in terms of relatively highvs low (for example) are maintained. Similarly, in some embodiments,protein levels can be used for one marker, while a second marker can beanalyzed via DNA, and, for example, a third marker can be analyzed viamRNA. Thus, the nature of the molecule being tested can be alteredwithin a test, if desired.

In some embodiments, the level of expression is determined by detectingthe level of mRNA transcribed from a gene.

In some embodiments, the mRNA in the sample is first transcribed intocDNA using reverse transcriptase.

In some embodiments, the sample is subjected to an amplificationreaction (e.g., using methods based on polymerase chain reaction (PCR),nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), strand displacementamplification (SDA), etc.), probe amplification (e.g., using methodsbased on ligase chain reaction (LCR), cleavase invader, etc.), signalamplification (e.g, using methods based on branched DNA probes [bDNA],hybrid capture, etc.), and others as will be readily understood by thoseof skill in the art.

In some embodiments, the mRNA is detected in the sample by hybridizing anucleic acid probe or primer capable of selectively hybridizing to amRNA transcript of interest, or cDNA derived therefrom, and thendetecting the hybridization with a detection device or system.

In this context, the term “selective hybridization” means thathybridization of a probe or primer occurs at a higher frequency or rate,or has a higher maximum reaction velocity, than hybridization of thesame probe or primer to any other nucleic acid. Preferably, the probe orprimer does not hybridize to another nucleic acid at a detectable levelunder the reaction conditions used.

As transcripts of a gene described herein are detected using mRNA orcDNA derived therefrom, assays that detect changes in mRNA can beemployed (for example, Northern hybridization, RT-PCR, NASBA, TMA orligase chain reaction).

In some embodiments, mRNA quantitation can be carried out on geneexpression array platforms, including Agilent's Bioanalyzer® system;Affymetrix′ GeneChip®, GeneTitan®, or GeneAtlas® systems; and others aswill be readily understood by those of skill in the art.

In some embodiments, mRNA quantitation can be carried out by real-timeqPCR. Such reactions can be performed with a variety of reporters,including non-specific DNA-binding fluorochromes (e.g., SYBR® Green) orfluorescent reporter probes that selectively hybridize to the sequenceof interest (e.g., TaqMan® probes). In some cases, the reaction iscarried out in parallel on multiple partitions of the same sample(digital PCR). Real-time qPCR platforms include ThermoFisherScientific/Applied Biosystems' FAST, QuantStudio, and related systems;Hologic/Gen-Probe's DTS systems; Roche/Idaho Technology's LightCycler®systems; Qiagen's Rotor-Gene® systems; Bio-Rad's CFX and relatedsystems; and others as will be readily understood by those of skill inthe art.

In some embodiments, mRNA quantitation can be carried out without areverse-transcription or amplification step, such as NanoString's invitro nCounter® platform, or various in situ hybridization (ISH)approaches, including paired probe ISH (e.g., RNAscope® and Quanti-GeneRNAview), single-tag multi-probe ISH (e.g., Stellaris®), or lockednucleic-acid (LNA) probes.

In some embodiments, mRNA quantitation can be carried out with RNA-Seqnext-generation sequencing technologies, including sequencing bysynthesis (e.g, Illumina's HiSeq® and NextSeq® systems), single-moleculereal-time sequencing (e.g., Pacific Biosciences' RS systems), ionseminconductor sequencing (e.g., ThermoFisher Scientific's Ion Torrent™systems), sequencing by ligation (e.g., ThermoFisher Scientific's SOLiD™systems), pyrosequencing (e.g., Roche/454 Life Sciences), and others aswill be readily understood by those of skill in the art.

Methods of RT-PCR include, for example, in Dieffenbach (ed) and Dveksler(ed) (In: PCR Primer: A Laboratory Manual, Cold Spring HarbourLaboratories, N Y, 1995). Essentially, this method comprises performinga PCR reaction using cDNA produced by reverse transcribing mRNA from acell using a reverse transcriptase. Methods of PCR described supra areto be taken to apply mutatis mutandis to this embodiment of theinvention.

Similarly PCR can be performed using cDNA. One or more of the probes orprimers used in the reaction specifically hybridize to the transcript ofinterest.

Methods of TMA or self-sustained sequence replication (3 SR) use two ormore oligonucleotides that flank a target sequence, a RNA polymerase,RNase H and a reverse transcriptase. One oligonucleotide (that alsocomprises a RNA polymerase binding site) hybridizes to an RNA moleculethat comprises the target sequence and the reverse transcriptaseproduces cDNA copy of this region. RNase H is used to digest the RNA inthe RNA-DNA complex, and the second oligonucleotide used to produce acopy of the cDNA. The RNA polymerase is then used to produce a RNA copyof the cDNA, and the process repeated.

NASBA systems relies on the simultaneous activity of three enzymes (areverse transcriptase, RNase H and RNA polymerase) to selectivelyamplify target mRNA sequences. The mRNA template is transcribed to cDNAby reverse transcription using an oligonucleotide that hybridizes to thetarget sequence and comprises a RNA polymerase binding site at its 5′end. The template RNA is digested with RNase H and double stranded DNAis synthesized. The RNA polymerase then produces multiple RNA copies ofthe cDNA and the process is repeated.

In some embodiments, a microarray can be used to determine the level ofexpression of one or more nucleic acids described herein. Such a methodallows for the detection of a number of different nucleic acids, therebyproviding a multi-analyte test and improving the sensitivity and/oraccuracy of the diagnostic assay of the invention.

In some embodiments, the level of expression is determined by detectingthe level of a protein encoded by a nucleic acid within a gene describedherein.

In this respect, the embodiments are not necessarily limited to thedetection of a protein comprising the specific amino acid sequencerecited herein. Rather, the present invention encompasses the detectionof variant sequences (e.g., having at least about 80% or 90% or 95% or98% amino acid sequence identity) or the detection of an immunogenicfragment or epitope of said protein.

The amount, level and/or presence of a polypeptide can be determinedusing any of a variety of techniques known to the skilled artisan suchas, for example, a technique selected from the group of,immunohistochemistry, immunofluorescence, an immunoblot, a Western blot,a dot blot, an enzyme linked immunosorbent assay (ELISA),radioimmunoassay (MA), enzyme immunoassay, fluorescence resonance energytransfer (FRET), matrix-assisted laser desorption/ionization time offlight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry(including tandem mass spectrometry, e.g. LC MS/MS), biosensortechnology, evanescent fiber-optics technology or protein chiptechnology.

In some embodiments, the assay used to determine the amount or level ofa protein is a semi-quantitative assay. In some embodiments, the assayused to determine the amount or level of a protein in a quantitativeassay. As will be apparent from the preceding description, such an assaymay involve the use of a suitable control, e.g. from a normal individualor matched normal control.

In some embodiments, standard solid-phase ELISA or FLISA formats can beuseful in determining the concentration of a protein from a variety ofsamples.

In one form such an assay involves immobilizing a biological sample ontoa solid matrix, such as, for example a polystyrene or polycarbonatemicrowell or dipstick, a membrane, or a glass support (e.g. a glassslide). An antibody that specifically binds to a protein describedherein is brought into direct contact with the immobilized biologicalsample, and forms a direct bond with any of its target protein presentin said sample. This antibody is generally labeled with a detectablereporter molecule, such as for example, a fluorescent label (e.g. FITCor Texas Red) or a fluorescent semiconductor nanocrystal (as describedin U.S. Pat. No. 6,306,610) in the case of a FLISA or an enzyme (e.g.horseradish peroxidase (HRP), alkaline phosphatase (AP) orbeta-galactosidase) in the case of an ELISA, or alternatively a secondlabeled antibody can be used that binds to the first antibody. Followingwashing to remove any unbound antibody the label is detected eitherdirectly, in the case of a fluorescent label, or through the addition ofa substrate, such as for example hydrogen peroxide, TMB, or toluidine,or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (x-gal) in the caseof an enzymatic label.

In some embodiments, an ELISA or FLISA comprises immobilizing anantibody or ligand that specifically binds a protein described supra ona solid matrix, such as, for example, a membrane, a polystyrene orpolycarbonate microwell, a polystyrene or polycarbonate dipstick or aglass support. A sample is then brought into physical relation with saidantibody, and the polypeptide is bound or ‘captured’. The bound proteinis then detected using a labeled antibody. For example, a labeledantibody that binds to an epitope that is distinct from the first(capture) antibody is used to detect the captured protein.Alternatively, a third labeled antibody can be used that binds thesecond (detecting) antibody.

In some embodiments, the presence or level of a protein is detected in abody fluid using, for example, a biosensor instrument (e.g., BIAcore™,Pharmacia Biosensor, Piscataway, N.J.). In such an assay, an antibody orligand that specifically binds a protein is immobilized onto the surfaceof a receptor chip. For example, the antibody or ligand is covalentlyattached to dextran fibers that are attached to gold film within theflow cell of the biosensor device. A test sample is passed through thecell. Any antigen present in the body fluid sample, binds to theimmobilized antibody or ligand, causing a change in the refractive indexof the medium over the gold film, which is detected as a change insurface plasmon resonance of the gold film.

In some embodiments, the presence or level of a protein or a fragment orepitope thereof is detected using a protein and/or antibody chip. Toproduce such a chip, an antibody or ligand that binds to the antigen ofinterest is bound to a solid support such as, for example glass,polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, goldor silicon nitride. This immobilization is either direct (e.g. bycovalent linkage, such as, for example, Schiff s base formation,disulfide linkage, or amide or urea bond formation) or indirect.

To bind a protein to a solid support it is often useful to treat thesolid support so as to create chemically reactive groups on the surface,such as, for example, with an aldehyde-containing silane reagent or thecalixcrown derivatives described in Lee et al, Proteomics, 3: 2289-2304,2003. A streptavidin chip is also useful for capturing proteins and/orpeptides and/or nucleic acid and/or cells that have been conjugated withbiotin (e.g. as described in Pavlickova et al., Biotechniques, 34:124-130, 2003). Alternatively, a peptide is captured on amicrofabricated polyacrylamide gel pad and accelerated into the gelusing microelectrophoresis as described in, Arenkov et al. Anal.Biochem. 278:123-131, 2000.

Other assay formats are also contemplated, such as flow-throughimmunoassays (PCT/AU2002/01684), a lateral flow immunoassay(US20040228761, US20040248322 or US20040265926), a fluorescencepolarization immunoassay (FPIA) (U.S. Pat. Nos. 4,593,089, 4,492,762,4,668,640, and 4,751,190), a homogeneous microparticles immunoassay(“HMI”) (e.g., U.S. Pat. Nos. 5,571,728, 4,847,209, 6,514,770, and6,248,597) or a chemiluminescent microparticle immunoassay (“CMIA”). Thecontents of all of the patent applications and patents disclosed in thisparagraph are incorporated herein by reference in their entirety. Insome embodiments, mRNA can be used to assay for HER2. In someembodiments, mRNA is not used for assaying for SIAH2.

In some embodiments, “elevated risk” for DCIS is defined as thatdetailed in Gorringe and Fox, Ductal Carcinoma In Situ Biology,Biomarkers, and Diagnosis, Frontiers in Oncology, Vol. 71-14, October2017, the entirety of which is hereby incorporated by reference. In someembodiments, any one or more of the conditions, markers, etc. noted inGorringe and Fox can be used to define the elevated risk aspect providedand employed herein in initially identifying a subject as one in theelevated risk grouping, and from which one then continues on with theother testing, analysis, and/or treatment options provided herein. Insome embodiments, “elevated risk” for DCIS is defined as an elevatedrisk under a DCISionRT analysis. In some embodiments, the DCISionRTanalysis is that described in Bremer et al., “A Biologic Signature forBreast Ductal Carcinoma in situ to Predict Radiation Therapy benefit andAssess Recurrence Risk”, American Association of Cancer Research, inClinical Cancer Research, doi: 10.1158/1078-0432.CCR-18-0842, Jul. 27,2018, the entirety is incorporated herein by reference. In someembodiments, elevated risk (for DCIS recurrence or invasive breastcancer in a subject with DCIS) is as determined by any one or more ofthe compositions or techniques in U.S. Pat. Pub. No. 2017/0350895, theentirety of which is hereby incorporated by reference.

Treatments by Test Results

For subjects at risk of a subsequent invasive ipsilateral breast event,then the subject may be treated with at least BCS plus radiation therapy(RT), and may receive further adjuvant therapy of at least hormonetherapy (e.g., tamoxifen or an aromase inhibitor), and/or HER2 therapy(e.g., Trastuzumab). The selection of which option will depend uponwhether or not the subject will respond to radiation therapy, based onthe present disclosure (e.g., elevated k-ras members and/or HER2+ andSIAH+). In some embodiments, if a subject has a high likelihood of bothan invasive ipsilateral breast event and a DCIS ipsilateral breast eventthen the subject can be treated with mastectomy and can receive furtheradjuvant therapy of at least hormone therapy and/or HER2 therapy, if thesubject will not respond to radiation therapy (e.g., elevated k-raspathway and/or HER2+ and SIAH+).

In some embodiments, rather than relying on standard clinical andpathologic factors to determine treatment or a risk profile tointerpret, one can use a signature of biomarkers to direct performanceof a therapy. This ability allows one a superior approach to therapywithout having to determine therapy after interpreting a risk profile.That is, as compared to current DCIS standard of care (breast-conservingsurgery (BCS) with adjuvant radiation therapy), more appropriatetreatment may be given to patients according to a signature recommendingtreatment for patients based on a biological profile detailed anddescribed in Tables 13-15 and included herein by example. In someembodiments, a subject is treated with BCS or frequent breast imagingfor early detection of an ipsilateral breast event (watchful waiting) toreduce likelihood of a subsequent ipsilateral breast event. In someembodiments, the guided treatment is at least BCS plus radiation therapyand may include further adjuvant therapy of at least hormone therapy(e.g., tamoxifen or an aromatase inhibitor), and/or HER2 therapy (e.g.,Trastuzumab) to reduce the likelihood of an invasive ipsilateral breastevent. In some embodiments, if the subject is SIAH+ and HER2+ and/or hasan elevated k-ras pathway, then radiation therapy is not employed andinstead HER2 therapy can be employed. In some embodiments, the subjectcan be treated with mastectomy and can receive further adjuvant therapyof at least hormone therapy and/or HER2 therapy to reduce the likelihoodthat a subject/patient has an invasive ipsilateral breast event and/or aDCIS ipsilateral breast event (especially when the subject is refractoryto radiation therapy).

TABLE 13 (INVASIVE RISK) EXAMPLE RISK ALTERNATIVE NUMBER LEVELCOMPONENTS TREATMENT TREATMENT 1) LOWER PR is POSITIVE and (AGE is LOWERRISK LOWER RISK RISK LOW) TREATMENT TREATMENT ELEVATED PR is NEGATIVEand (AGE is MASTECTOMY BCS + RT + RISK LOW) ADJUVANT THERAPY 2) LOWER(PR is POSITIVE and HER2 is LOWER RISK LOWER RISK RISK NEGATIVE) and(AGE is LOW) TREATMENT TREATMENT ELEVATED (PR is NEGATIVE OR HER2 isMASTECTOMY BCS + RT + RISK POSITIVE) and (AGE is LOW) ADJUVANT THERAPY3) LOWER (PR is POSITIVE and HER2 is LOWER RISK LOWER RISK RISKNEGATIVE) and (AGE is LOW) TREATMENT TREATMENT ELEVATED (HER2 isPOSITIVE) and (AGE is BCS + RT, MASTECTOMY RISK LOW) ADJUVANT HER2TREATMENT ELEVATED (HER2 IS NEGATIVE and PR is MASTECTOMY BCS +RT RISKNEGATIVE) and (AGE is LOW) 4) LOWER (SIAH2 is HIGH or HER2 is LOWER RISKLOWER RISK RISK POSITIVE) and (PR is NEGATIVE) TREATMENT TREATMENTELEVATED (SIAH2 is LOW and HER2 is BCS + RT RISK NEGATIVE ) and (PR isNEGATIVE) 5) LOWER (FOXA1 is ELEVATED or HER2 is LOWER RISK LOWER RISKRISK POSITIVE ) and (PR is TREATMENT TREATMENT NEGATIVE) ELEVATED FOXA1IS LOW and HER2 is BCS + RT BCS + RT RISK NEGATIVE and (PR is NEGATIVE)6) LOWER (SIAH2 is ELEVATED and LOWER RISK LOWER RISK RISK FOXA1 isELEVATED and HER2 TREATMENT TREATMENT is NEGATIVE) and (AGE is ELEVATEDELEVATED and HER2 is POSITIVE) and PR is NEGATIVE ELEVATED (SIAH2 is LOWor FOXA1 is BCS + RT MASECTOMY RISK LOW) and HER2 is NEGATIVE and PR isNEGATIVE) or (AGE is LOW and HER2 is POSITIVE and PR is NEGATIVE) 7)LOWER (FOXA1 is LOW or HER2 is LOWER RISK LOWER RISK PR RISK POSITIVE)and AGE ELEVATED TREATMENT TREATMENT POSITIVE and PR is POSITIVEELEVATED FOXA1 is ELEVATED and HER2 BCS + RT RISK is NEGATIVE) and AGEELEVATED and PR is POSITIVE TABLE 13 LEGEND: FOXA1 is LOW FOXA1 ASSESSEDBY IMMUNOSCORE <150 IMMUNOSCORE (INTENSITY TIMES PERCENTAGE) FOXA1 isFOXA1 ASSESSED BY IMMUNOSCORE >=150 IMMUNOSCORE ELEVATED (INTENSITYTIMES PERCENTAGE) PR is NEGATIVE PR ASSESSED BY PERCENTAGE <15% PR isPOSITIVE PR ASSESSED BY PERCENTAGE >=15% HER2 is HER2 ASSESSED BY IHC AS(EITHER 1+ OR 2+) HER2 IHC (1+ OR 2+), OR NEGATIVE OR BY FISH ASNEGATIVE OR BY SISH AS FISH NEG, OR SISH NEG; NEGATIVE; HOWEVER IF ANYOF THESE ARE HOWEVER IF ANY OF POSITIVE THEN THE RESULT IS POSITIVETHESE ARE POSITIVE THEN THE RESULT IS POSITIVE HER2 is POSITIVE HER2ASSESSED BY IHC AS (EITHER 3+) OR HER2 IHC (3+), OR FISH BY FISH ASPOSITIVE OR BY SISH AS POS, OR SISH POS; POSITIVE HOWEVER IF ANY OF AREPOSITIVE WHERE IF ANY OF POSITIVE THEN THE RESULT IS POSITIVE THESE ARETHESE THEN THE RESULT IS POSITIVE AGE is LOW AGE assessed by patient ageat diagnosis of initial AGE <50 DCIS AGE is AGE assessed by patient ageat diagnosis of initial AGE >=50 ELEVATED DCIS SIAH2 is LOW SIAH2ASSESSED BY IHC AS PERCENTAGE <20 SIAH2 is SIAH2 ASSESSED BY IHC ASPERCENTAGE >=30 ELEVATED PALPABLE The patient presented following thefinding of a IS PALPABLE palpable mass in the breast or a physical examfound a palpable mass in the breast NOT PALPABLE The patient was notfound to have a palpable mass in NOT PALPABLE the breast

TABLE 14 (DCIS RISK) REFERENCE EXAMPLE RISK ALTERNATIVE TABLE IN NUMBERLEVEL COMPONENTS TREATMENT TREATMENT APPLICATION 1) LOWER SIAH2 is LOWand AGE LOWER DCIS LOWER RISK 5 RISK is ELEVATED RISK TREATMENTTREATMENT ELEVATED SIAH2 is ELEVATED BCS + RT RISK and AGE is ELEVATEDLOWER DCIS LOWER RISK 3, 5 2) LOWER SIAH2 is LOW and AGE RISK TREATMENTRISK is ELEVATED TREATMENT ELEVATED SIAH2 is ELEVATED ADJUVANT BCS + RTRISK BCS + RT + HORMONE and PR is POSITIVE and TREATMENT AGE is ELEVATEDELEVATED SIAH2 is ELEVATED BCS + RT RISK and PR is NEGATIVE and AGE isELEVATED 3) LOWER SIAH2 is LOW and AGE LOWER DCIS LOWER RISK 2, 5 RISKis ELEVATED RISK TREATMENT TREATMENT ELEVATED SIAH2 is ELEVATED BCS + RTBCS + RT + RISK and HER2 is POSITIVE ADJUVANT and AGE is TARGETEDELEVATED HER2 TREATMENT ELEVATED SIAH2 is ELEVATED BCS + RT RISK andHER2 is NEGATIVE and AGE is ELEVATED 4) LOWER SIAH2 is LOW and ( PRLOWER DCIS LOWER RISK 4 RISK is NEGATIVE) RISK TREATMENT TREATMENTELEVATED SIAH2 is ELEVATED BCS + RT RISK and (PR is NEGATIVE or HER2 isPOSITIVE) 5) LOWER SIAH2 is LOW and ( PR LOWER DCIS LOWER RISK 4 RISK isNEGATIVE) RISK TREATMENT TREATMENT ELEVATED SIAH2 is ELEVATED BCS + RTBCS + RT + RISK and HER2 is POSITIVE ADJUVANT TARGE IED HER2 TREATMENTELEVATED SIAH2 is ELEVA 1 ED BCS + RT RISK and PR is NEGATIVE and HER2is NEGATIVE 6) LOWER SIAH2 is LOW and ( PR LOWER DCIS LOWER RISK 4, 5RISK is NEGATIVE) RISK TREATMENT TREATMENT ELEVATED SIAH2 is ELEVATEDBCS + RT RISK and ( PR is NEGATIVE or HER2 is POSITIVE or AGE isELEVATED) 7) LOWER SIAH2 is LOW and ( PR LOWER DCIS LOWER RISK 4, 5 RISKis NEGATIVE) RISK TREATMENT TREATMENT ELEVATED SIAH2 is ELEVATED BCS +RT BCS + RT + RISK and HER2 is POSITIVE ADJUVANT TARGETED HER2 TREATMENTELEVATED SIAH2 is ELEVATED BCS + RT RISK and ( PR is NEGATIVE or AGE isELEVATED ) and HER2 is NEGATIVE LOWER DCIS LOWER RISK 6 8) LOWER FOXA1is ELEVATED RISK TREATMENT RISK and ( PR is POSITIVE) TREATMENT ELEVATEDFOXA1 is LOW and PR BCS + RT BCS + RT + RISK is POSITIVE ADJUVANTHORMONE TREATMENT 9) LOWER ( (FOXA1 is LOWER DCIS LOWER RISK 9 RISKELEVATED and PR is RISK TREATMENT POSITIVE) or PR is TREATMENT NEGATIVE) and (SIAH2 is LOW and (PR is NEGATIVE or HER2 is POSITIVE) ) ELEVATED(FOXA1 is LOW and PR BCS + RT BCS + RT + RISK is POSITIVE ) OR (ADJUVANT(SIAH2 is ELEVA I ED HORMONE AND (PR is NEGATIVE TREATMENT if or HER2 isPOSITIVE) ) PR is POS) or (ADJUVANT TARGETED HER2 TREATMENT if HER2 isPOS) 10) LOW RISK ( (FOXA1 is LOWER DCIS LOWER RISK 4, 5, 8, 9 ELEVATEDand PR is RISK TREATMENT POSITIVE) or PR is TREATMENT NEGATIVE ) and(SIAH2 is LOW and (PR is NEGATIVE or HER2 is POSITIVE or AGE isELEVATED) ) ELEVATED (SIAH2 is ELEVATED BCS + RT RISK and ( PR isNEGATIVE or HER2 is POSITIVE or AGE is ELEVATED) ) or (FOXA1 is LOW andPR is POSITIVE) TABLE 14 LEGEND: “<” means less than “<=” means lessthan or equal to “>” means greater than “>=” means greater than or equalto FOXA1 is LOW FOXA1 ASSESSED BY IMMUNOSCORE <150 IMMUNOSCORE(INTENSITY TIMES PERCENTAGE) FOXA1 is FOXA1 ASSESSED BYIMMUNOSCORE >=150 IMMUNOSCORE ELEVATED (INTENSITY TIMES PERCENTAGE) PRis PR ASSESSED BY PERCENTAGE <15% NEGATIVE PR is POSITIVE PR ASSESSED BYPERCENTAGE >=15% HER2 is HER2 ASSESSED BY IHC AS (EITHER 1+ HER2 IHC (1+OR 2+), OR FISH NEGATIVE OR 2+) OR BY FISH AS NEGATIVE OR NEG, OR SISHNEG; HOWEVER BY SISH AS NEGATIVE; HOWEVER IF IF ANY OF THESE ARE ANY OFTHESE ARE POSITIVE THEN POSITIVE THEN THE RESULT IS THE RESULT ISPOSITIVE POSITIVE HER2 is HER2 ASSESSED BY IHC AS (EITHER HER2 IHC (3+),OR FISH POS, POSITIVE 3+) OR BY FISH AS POSITIVE OR BY SISH AS POSITIVEHOWEVER IF ANY OR SISH POS; WHERE IF ANY OF THESE ARE POSITIVE THEN THEOF THESE ARE POSITIVE THEN RESULT IS POSITIVE THE RESULT IS POSITIVE AGEis LOW AGE assessed by patient age at diagnosis of AGE <50 initial DCISAGE is AGE assessed by patient age at diagnosis of AGE >=50 ELEVATEDinitial DCIS SIAH2 is LOW SIAH2 ASSESSED BY IHC AS <20 PERCENTAGE SIAH2is SIAH2 ASSESSED BY IHC AS >=30 ELEVATED PERCENTAGE PALPABLE Thepatient presented following the finding of IS PALPABLE a palpable massin the breast or a physical exam found a palpable mass in the breast NOTPALPABLE The patient was not found to have a palpable NOT PALPABLE massin the breast LOWER DCIS RISK TREATMENT: Breast conserving surgery (BCS)ONLY, without adjuvant therapy or BCS with adjuvant hormone treatment,in combination with either standard or more frequent than standardsurveillance may be selected for patients with LOWER DCIS RISK. However,for patients assessed as having LOWER DCIS RISK of recurrence, thetreatment also needs to reflect the risk potential for subsequentinvasive breast cancer. Additionally, the patient risk tolerance may beconsidered in selecting the nature of the treatment. For example, ifDCIS recurrence is evaluated as LOWER RISK, but the risk of INVASIVEbreast cancer after initial diagnosis of DCIS is ELEVATED RISK, thenbreast conserving surgery with (adjuvant radiation therapy, adjuvanttargeted HER2 treatment, or adjuvant hormone treatment), or mastectomywould be selected. For example, if DCIS recurrence is evaluated as LOWERRISK, but the risk of INVASIVE breast cancer after initial diagnosis ofDCIS is UNKNOWN, then breast conserving surgery with (adjuvant radiationtherapy, adjuvant targeted HER2 treatment, or adjuvant hormonetreatment), or mastectomy would be selected. In the case that the riskof recurrence of DCIS is ELEVATED RISK, then Breast Conserving Surgerywith Radiation therapy would be recommended. The patient risk tolerancemay be considered in the selection of the therapy for patients withELEVATED DCIS RISK. Adjuvant treatment consisting of adjuvant hormonetreatment or adjuvant targeted treatment for HER2 may be selected as thetreatment to augment breast conserving surgery alone.

In some embodiments, the marker signatures disclosed and describedherein can be used to comprehensively determine a total recurrence orprogression risk and/or the likelihood that a subject will respond orwill not respond to radiation therapy and therefore, determine a moreappropriate treatment. The (a) markers listed in Table 13 are used toassess risk of an invasive breast cancer and (b) the markers listed inTable 14 are used to assess risk of a DCIS event. In combination, thetotal risk, which includes risk of an invasive event and risk of a DCISevent, may be determined and used to recommend a more appropriatetreatment. For example, if a patient is found to be of low risk for aninvasive event according to Table 13 but at a high risk for a DCIS eventaccording to Table 14, the patient would be at a high total risk ofrecurrence. As disclosed and described herein, the various methods andcompositions can allow one to determine the likelihood of (a) arecurrent breast event, (b) progression to invasive or metastaticcancer, or (c) whether or not a subject/patient with DCIS now willexperience no further DCIS/invasive breast cancer, experience DCISand/or experience invasive breast cancer and to thus, treat a patientdiagnosed with DCIS in a more appropriate manner. The same can beapplied for Table 15, as a combined option. Then, one can furtherdetermine, based on for example, the k-ras pathway levels and/or HER2and SIAH2 state of the sample, if the subject will be responsive toradiation therapy, and thereby determine if the subject should receiveradiation therapy or a non-radiation therapy such as a HER2 antibody,such as trastuzumab. Thus, in some embodiments, any option fordetermining risk level can be combined with a further option (whereappropriate) for determining k-ras pathway levels and/or HER2 and SIAH2state of the sample to determine if the subject will be responsive toradiation therapy, and thereby determine if the subject should receiveradiation therapy or a non-radiation therapy such as a HER2 antibody,such as trastuzumab (as detailed herein).

In some embodiments, the marker signatures disclosed and describedherein can be used to comprehensively treat a patient in a manner thatappropriately reduces the risk of total recurrence or progression,including an invasive event and/or a DCIS event. For example, (a) themarker signatures in Table 13 can be used to identify a treatment thatreduces the risk of an invasive event and (b) the marker signatures inTable 14 can be used to identify a treatment that reduces the risk of anDCIS event and/or identify treatment to reduce total risk according toTable 13 and Table 14. The same can be applied for Table 15, as acombined option. The subject at elevated risk of an invasive event canthen be treated with either a radiation or non-radiation therapy, asoutlined herein.

The following is a non-exhaustive list of examples of treatmentalternatives for patients based on their individual risk profiles as setforth in Tables 13-15.

Example 1

A DCIS sample from a subject having DCIS is analyzing for SIAH2 andHER2, and for at least one of PR, FOXA1, or (in the alternative)analyzed for FOXA1 and PR. The results are compared with the matrix inTables 9 and/or 11 to determine if the subject has an elevated risk ofinvasive breast cancer, DCIS recurrence, or neither. If the subject hasan elevated risk of invasive breast cancer, one reviews the SIAH2 andHER2 levels. A subject that is HER2+ and SIAH2+ will not receiveradiation therapy and will instead receive trastuzumab.

Example 2

A DCIS sample from a subject is analyzed for a level of at least PR,HER2 and SIAH2, or (in the alternative) analyzing the sample for atleast FOXA1. A prognosis is provided based upon at least PR, HER2 andSIAH2 or based upon at least PR and FOXA1. Depending upon the nature ofthe results, this indicates that the subject that provided the sample isat a high or elevated risk of invasive breast cancer (see, e.g., Tables9 and/or 11). If the subject has an elevated risk of invasive breastcancer, one reviews the SIAH2 and HER2 levels. A subject that is HER2+and SIAH2+ will not receive radiation therapy and will instead receivean anti-HER2 antibody.

Example 3

A DCIS sample from a subject is analyzed for a level of at least SIAH2and FOXA1. A prognosis is provided with the subject having an elevatedrisk of an invasive breast cancer based upon the level of at least SIAH2and FOXA1. If the subject has an elevated risk of invasive breastcancer, one reviews the SIAH2 and HER2 levels. A subject that is HER2+and SIAH2+ will not receive radiation therapy and will instead receivean anti-HER2 antibody.

Example 4

A DCIS sample from a subject is analyzed for: a) PR, HER2, and SIAH2, or(in the alternative) b) PR and FOXA1. A prognosis is provided for thesubject as having an elevated risk of an invasive breast cancer eventwhen at least one of: a) PR−, HER2−, and SIAH2−, b) PR+, FOXA1+, or c)PR+, FOXA1−, and Ki67+. If the subject has an elevated risk of invasivebreast cancer, one reviews the SIAH2 and HER2 levels. A subject that isHER2+ and SIAH2+ will not receive radiation therapy and will insteadreceive an anti-HER2 antibody.

Example 5

A DCIS sample is analyzed for at least one of: a) SIAH2 and FOXA1, b)SIAH2 and at least one of i) PR and ii) HER2, c) SIAH2 andpost-menopausal status; or (in the alternative) d) PR and FOXA1. Aprognosis is provided that the subject has an elevated risk of a DCISevent when at least one of: a) i) SIAH2+ and FOXA1+, b) SIAH2+ and HER2+or PR−; SIAH2+ and post-menopausal; or PR+ and FOXA1−, is present in theDCIS sample. If the subject has an elevated risk of invasive breastcancer, one reviews the SIAH2 and HER2 levels. A subject that is HER2+and SIAH2+ will not receive radiation therapy and will instead receivean anti-HER2 antibody.

Example 6

A DCIS sample is analyzed for at least one of: a) PR−, HER2—, andSIAH2−, b) PR+, FOXA1+, or c) PR+, FOXA1−, and Ki67+. When the analysisindicates a high risk of invasive breast cancer (see, Tables 9 and/or11) the subject is administered a therapy that is more aggressive thanstandard of care for DCIS. If the subject has an elevated risk ofinvasive breast cancer, one reviews SIAH2 and HER2 levels. A subjectthat is HER2+ and SIAH2+ will not receive radiation therapy and willinstead receive an anti-HER2 antibody.

Example 7

A DCIS sample is analyzed for at least one of: a) SIAH2+ and FOXA1+, b)SIAH2+ and HER2+ or PR−, c) SIAH2+ and post-menopausal status, or (inthe alternative) d) PR+ and FOXA1-. When the analysis indicates a highlikelihood of an invasive breast cancer, one administers to the subjecta more aggressive therapy than standard of care for a single DCIS event.If the subject has an elevated risk of invasive breast cancer, onereviews the SIAH2 and HER2 levels. A subject that is HER2+ and SIAH2+will not receive radiation therapy and will instead receive an anti-HER2antibody.

Example 8

A subject provides a sample that is analyzed for at least one of thecombinations as outlined in any one of Tables 13-15 to provide a risklevel for the subject. A corresponding treatment is then administered tothe subject, as outlined in the appropriate row in Tables 13-15, basedon the indicated risk level for the subject. If the subject has anelevated risk of invasive breast cancer, one reviews the SIAH2 and HER2levels. A subject that is HER2+ and SIAH2+ will not receive radiationtherapy and will instead receive an anti-HER2 antibody.

Example 9

As shown in FIG. 1, the greater majority of subjects that have DCIS,that are at an elevated risk of invasive breast cancer receive asignificant benefit from radiation therapy. However, as shown in FIG. 2,there is a non-responsive subgrouping of these people who do not respondto radiation therapy. These individuals are HER2+ and SIAH+.

1. A method of treating a subject, the method comprising: identifying asubject with DCIS that has an elevated level of activity in a k-raspathway; and administering an aggressive breast cancer therapy to thesubject.
 2. The method of claim 1, wherein the k-ras pathway is elevatedif there is an elevated level of at least one of: K-ras, RAF, MAPK, MEK,ETS or SIAH, and wherein the subject is HER2 positive.
 3. A method oftreating a subject, the method comprising: identifying a subject withDCIS, that is HER2 positive and SIAH2 positive; and administering anaggressive breast cancer therapy to the subject, wherein the aggressivebreast cancer therapy is aggressive radiation therapy or aggressivenon-radiation therapy.
 4. (canceled)
 5. A method of identifying asubject for an aggressive cancer therapy, the method comprising:identifying a subject with DCIS at an elevated risk of invasive breastcancer; and determining if the subject is HER2 and SIAH2 positive,wherein if the subject is HER2 and SIAH2 positive, administering anaggressive therapy to the subject, wherein the aggressive therapy: a) isnot radiation therapy; b) is aggressive radiation therapy; c) ischemotherapy; d) is a therapy that comprises an anti-HER2 antibody; ore) is selected from the group consisting of: an antibody to HER2 orTrastuzumab.
 6. (canceled)
 7. A method for treating a subject, saidmethod comprising: providing a DCIS sample from a subject; analyzing theDCIS sample for a level of at least PR and HER2, and at least either: a)analyzing the sample for at least SIAH2, or b) analyzing the sample forat least FOXA1; and providing a prognosis based upon at least PR, HER2and SIAH2 or based upon at least PR and FOXA1, wherein if the sample isPR positive, further analyzing the sample for a level of COX2, whereinCOX2 positive with at least FOXA1 positive indicates a high risk ofinvasive breast cancer, determining if the subject is HER2 positive; andadministering an aggressive therapy to the subject if the subject isHER2 positive, wherein the aggressive therapy is more aggressive thanthe standard of care therapy.
 8. The method of claim 3, furthercomprising: analyzing the sample for a level of Ki67; and identifyingthe subject as having an elevated risk of invasive breast cancer basedon a determination that there is a high level of Ki67 in the sample. 9.The method of claim 1, wherein the aggressive therapy comprises ananti-HER2 antibody.
 10. The method of claim 9, wherein the antibody isTrastuzumab.
 11. A method of providing a benefit of radiation therapy,the method comprising: identifying a subject with DCIS at elevated riskof invasive breast cancer; and administering radiation therapy to thesubject if the subject is HER2 negative and administering aggressiveradiation therapy or aggressive non-radiation therapy to the subject ifthe subject is HER2 positive. 12.-17. (canceled)
 18. The method of claim5, wherein the subject is high risk if they are PR positive and there isa very high level of FOXA1.
 19. The method of claim 5, wherein if asample is PR positive, then further analyzing the sample for Ki67, size,or both Ki67 and size.
 20. The method of claim 5, wherein if the sampleis PR positive, and wherein when FOXA1 negative, further analyzing thesample for a level of Ki67, size, or a level of Ki67 and size, whereinKi67 positive, a size larger than 5 mm of DCIS, or both, indicates highrisk.
 21. The method of claim 5, further comprising analyzing for p16,COX2, and Ki67.
 22. The method of claim 5, wherein analysis of eachmarker is carried out in parallel with each other.
 23. (canceled) 24.The method of claim 5, wherein PR analysis occurs first and any furtheranalysis depends upon the result of the PR analysis.
 25. The method ofclaim 5, wherein no additional markers are looked at to determine thetherapy.
 26. The method of claim 11, comprising: analyzing the samplefor a level of Ki67; and identifying the subject as having the elevatedrisk of invasive breast cancer based on a determination that there is ahigh level of Ki67 in the sample.
 27. A kit comprising: a firstdetectable binding agent specific for HER2; and a second detectablebinding agent specific for SIAH2.
 28. The kit of claim 27, furthercomprising one or more third detectable binding agents specific for oneor more of FOXA1, PR, Ki67, and p16.
 29. The kit of claim 27, whereinthe first and second detectable binding agents comprise a nucleic acidprobe or an antibody.